United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600 1-80-029
July 1980
Research and Development
Study of the Effect of
Whole Animal
Exposure to Acid
Mists & Particulates
on the Pulmonary
Metabolism of
Benzo(a)pyrene in the
Isolated Perfused
Lung Model
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-80-029
July 1980
STUDY OF THE EFFECT OF WHOLE ANIMAL EXPOSURE TO ACID MISTS AND
PARTICULATES ON THE PULMONARY METABOLISM OF BENZO(A)PYRENE IN
THE ISOLATED PERFUSED LUNG MODEL
By
Warshawsky, D., Niemeier, R.W.,* and
E. Bingham**
University of Cincinnati College of Medicine
Department of Environmental Health
3223 Eden Avenue
Cincinnati, Ohio 45267
*National Institute of Occupational Safety and Health
4676 Columbia Parkway
Mail Location C-23
Cincinnati, Ohio 45226
**0ccupational Safety and Health Administration
U.S. Department of Labor
200 Constitution Avenue
Washington, D.C. 20210
Contract No.: 68-02-1678
Final Report
Project Officer: Stephen Nesnow
Prepared for: Carcinogenesis and Metabolism Branch
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
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FOREWARD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance the
quality of our environment in order to promote the public health and welfare
and the productive capacity of our nation's population.
The Health Effects Research Laboratory, Research Triangle Park, conducts
a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data for
registration of new pesticides or proposed suspension of those already in
use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is pro-
vided in the form of expert testimony and preparation of affidavits as well
as expert advice to the Administrator to assure the adequacy of health care
and surveillance of persons having suffered imminent and substantial
endangerment of their health.
In this report, the metabolism of benzo(a)pyrene (BaP), an ubiquitous
environmental pollutant and proven carcinogen, in the lung is examined.
Because BaP must be metabolized to produce the carcinogenic response, an
understanding of its metabolism, and the potential inhabition of that
metabolism by other environmental pollutants (Fe20s,S02,CAP), is important
to the protection of human populations.
F. Gorden Hueter
Di rector
Health Effects Research Laboratory
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PREFACE
Our general interest in the lung originated from the fact that the
respiratory tract is the main portal of entry and one of the first surfaces
contacted by airborne contaminants. The main reason for our interest in
pulmonary disposition of pollutants is the potential importance of the
ultimate toxicity of some of these agents. Of interest, also, are the
agents, drugs, or pollutants reaching the lung via the circulatory system.
It has been well established that the lungs are capable of binding and/or
metabolizing several such agents.
There is no way to study the pulmonary metabolic activity in vivo
because of the influence of other organs. In vitro tissue preparations such
as slices and homogenates compromise the integrity of an investigation,
especially when considering concurrent administration of multiple agents in
different physical forms or when determining distribution or binding of
compounds throughout the pulmonary system. Therefore, the obvious choice in
our opinion was the isolated perfused lung (IPL).
A number of criteria were chosen and considered mandatory in order to
provide an isolated perfused lung preparation that was sufficiently stable
to permit evaluation of metabolic activity, distribution, and uptake of
compounds. In addition, we thought that monitoring of physiological and
biological indices would better define the stability of the system.
The system which was developed in this laboratory is relatively simple
to set up and not prohibitive in cost. The major features include:
1) undiluted heparinized autologous whole blood as the perfusate;
2) recirculation of the perfusate and thus accumulation of metabolites;
3) constant pressure perfusion, perfusate level being maintained in an
upper reservoir by electronic sensors; 4) chemically inert surfaces
(siliconized glass or silicone rubber tubing); 5) ventilation via
cycling subatmospheric pressure; 6) warming and humidification of ventila-
ting gas (air with C02); 7) regulation of blood pH through infusion of
sodium bicarbonate and adjustment of inhaled CQ2> 8) periodic monitoring of
biochemical and physiological conditions, including blood flow, respiratory
minute volume, blood gases, glucose uptake; lactate production, etc;
9) readily available sampling ports; 10) water-jacketed components; and
11) recovery of blood, lung washings, ventilating gas, trachea-bronchi, and
the remainder of respiratory tract.
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ABSTRACT
Lung cancer represents the highest single cause of cancer deaths in
the U.S. Epidemiological and experimental evidence indicates that the
interplay of multiple environmental factors is responsible for the induction
of lung cancer. Man is exposed to a complex mixture of potentially
hazardous materials, including specific carcinogens and a variety of agents
which may modify the manner in which the lung disposes of inhaled materials.
One such carcinogen is benzo(a)pyrene (BaP) a ubiquitous environmental
pollutant formed during the destructive distillation of coal and in other
processes that involve incomplete combustion of organic materials.
BaP in combustion with various agents, such as ferric oxide, has been
used in animals to experimentally induce tumors of bronchogenic origin.
Evidence describes the necessity for this compound, BaP, to be metabolized
to produce the carcinogenic response. However, the metabolism of BaP in
the lung has not been fully investigated.
Since at least three enzymes are involved in the metabolism of this
compound and some of these systems can be inhibited by the presence of
Fe203, S02, or CAP to produce different metabolic patterns, a study of all
the metabolites in the lung is necessary in order to determine if the rate
or pattern of formation has changed. Therefore, an isolated perfused rabbit
lung preparation suitable for metabolic studies has been developed in our
laboratory to study rate and pattern of formation of BaP in the presence of
crude air particulate and/or S02.
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TABLE OF CONTENTS
Page
Foreward iii
Preface iv
Abstract v
Table of Contents vi
Figures x
Tables xi
Abbreviations & Symbols xv
Acknowledgement xvi
A. Introduction 1
B. Conclusions 5
C. Recommendations 9
D. Materials and Methods 11
1. Chemicals 11
2. Instrumentation 12
3. Isolated Perfused Lung Preparation 12
a. Perfusion Apparatus 12
b. Preparation of Lungs 20
c. Start Up of Perfusion System 20
d. Cleaning the System 22
e. Modification 22
f. Pretreatment of BaP, CAP, & SMC 24
g. Administration of BaP & CAP to IPL 24
vi
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TABLE OF CONTENTS (continued)
Page
h. S02 Administration In Vivo and In Vitro 25
i. Extraction of Analysis of Biological Samples ... 29
E. Results 35
1. Effects of Enzyme Inducers 35
a. Influence of BaP Pretreatment 35
b. Influence of Various Pi450 Enzyme Inducers 43
c. Influence of PI 450 Enzyme Inducers on BaP
Metabolism 43
d. Distribution of BaP and Metabolites in Tissues at
180 Minutes in the IPL Following BaP
Pretreatment 43
Summary 44
2. Effects of Parti culates 54
a. Influence of Particulate Administered to IPL on
BaP Metabolism 54
1) Rate of Metabolism 54
2) Distribution of BaP and Its Metabolites in Tissue
at 180 Minutes 54
Summary 54
b. Influence of BaP Pretreatment and Particulate
Administered on IPL on BaP Metabolism 62
1) Rate of Metabolism 62
2) Distribution of BaP and Its Metabolites in Tissue
at 180 Minutes 62
Summary 72
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TABLE OF CONTENTS (continued)
c. Influence of Particulate Pretreatment on BaP
Metabolism . . ................. 72
1) Rate of Metabolism ........ . ....... 72
2) Distribution of BaP and Its Metabolites in Tissue
at 180 Minutes ................. 73
Summary ...................... 73
d. Influence of Crude Air Particulate on BaP
Metabolism ................... 84
1) Rate of Metabolism ................ 84
2) Distribution of BaP and Its Metabolites in
Tissue at 180 Minutes .............. 84
Summary ...................... 85
3. Effects of SO 2 .................... 94
a. Influence of S02 Pretreatment on BaP Metabolism . . 94
1) Rate of Metabolism ................ 94
2) Distribution of BaP and Its Metabolites in Tissue
at 180 Minutes ................. 94
Summary ...................... 106
b. Influence of S02 Administered to IPL on BaP
Metabolism ................... 107
1) Rate of Metabolism ................ 107
2) Distribution of BaP and Its Metabolites in
Tissue at 180 Minutes .............. 107
Summary ...................... 108
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TABLE OF CONTENTS (continued)
Page
c. Influence of S02 and CAP Administered to IPL on
BaP Metabolism 119
1) Rate of Metabolism 119
2) Distribution of BaP and Its Metabolites in Tissue
at 180 Minutes 119
Summary 129
d. HPLC Distribution Pattern for S02 Data 129
F. Discussion 136
1. Perfusion - Basic Requirements 136
2. Control Animals with BaP on IPL 142
3 Pertubations with IPL 143
4. Enzyme Inducers Effects 147
5. Particulate Effects 148
6. S02 Effects 150
References :.52
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FIGURES
Number Page
1 Composition of Crude Air Particulate 13
2 IPL Design 16
3 Simplified IPL Schematic 17
4 Cannulae for IPL 18
5 Solenoid Diagram 19
6 Tracheal Value Schematic 23
7 In Vitro - SC>2 Modification of IPL 27
8 In Vivo - S02 Modification of IPL 28
9 HPLC Chromatogram of BaP Standard using Varian Review
Phase Column - 10 u particle size, 25 cm x 2.2 mm 33
10 HPLC Chromatogram using HiBar II Reverse Phase Column
10 u particle size, 25 cm x 4.6 mm 34
11 Inadequate Blood Flow Rate 140
12 a. Blood Flow Rate After Addition of Heparin and Epinephrine. . . 141
b. Typical Blood flow Rate with BaP, Heparin and Epinephrine
Addition 141
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TABLES
Number Page
1 Major Features of Isolated Perfused Lung Preparation 4
2 Emission Spectrographic Analyses of Crude (air)
Particul ate Matter 14
3 BaP Standards on tic 32
4 Pretreatment Regimen of Enzyme Inducers 37
5 Influence of Enzyme Inducers on Total Metabolite
Appearance in the Blood 38
Influence of Enzyme Inducers on the Metabolism of Benzo(a)-
pyrene on the IPL (BaPIT and BaPjp)
6 Rate and Pattern of Metabolism in the Blood 39
7 Comparison of HPLC and tic Data 40
8 Comparison of HPLC and tic Data 41
9 Comparison of HPLC and tic Data 42
Influence of Enzyme Inducers on the Metabolism of Benzo(a)-
pyrene on the IPL (SMC and BaPjp)
10 Rate and Pattern of Metabolism in the Blood 45
11 Comparison of HPLC and tic Data 46
Influence of Enzyme Inducers on the Metabolism of Benzo(a)-
pyrene on the IPL (BaPTp and Corn Oil)
12 Rate and Pattern of Metabolism in the Blood 47
Enzyme Inducers
13 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes - S.E 48
14 % Distribution Pattern of BaP + Metabolites in Each Tissue 49
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TABLES (continued)
Number Page
Influence of Particulates Administered to IPL on BaP Metabolism
15 Rate and Pattern of Metabolism in the Blood 56
16 Comparison of HPLC and tic Data 57
17 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes - S.E 58
18 % Distribution Pattern of BaP + Metabolites in Each Tissue 59
Influence of BaP Pretreatment and Particulates Administered on IPL
on BaP Metabolism
19 Rate and Pattern of Metabolism in the Blood 64
20 Comparison of HPLC and tic Data 65
21 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes ± S.E 66
22 % Distribution Pattern of BaP + Metabolites in Each Tissue 67
Influence of Participate Pretreatment on BaP Metabolism
23 Rate and Pattern of Metabolism in the Blood 75
24 Comparison of HPLC and tic Data 76
25 Comparison of HPLC and tic Data 77
26 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes ± S.E 78
27 % Distribution Pattern of BaP + Metabolites in Each Tissue 79
Influence of Crude Air Particulate on BaP Metabolism
28 Rate and Pattern of Metabolism in the Blood 86
29 Comparison of HPLC and tic Data 87
30 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes - S.E 88
31 % Distribution Pattern of BaP + Metabolites in Each Tissue 89
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TABLES (continued)
Number Page
Influence of SO,, Pretreatment on BaP Metabolism
32a Rate and Pattern of Metabolism in the Blood 96
32b Rate and Pattern of Metabolism in the Blood 97
33 Comparison of HPLC and tic Data 98
34 Comparison of HPLC and tic Data "
35 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes * S.E 100
36 % Distribution Pattern of BaP + Metabolites in Each Tissue 101
Influence of S0? Administration to IPL on BaP Metabolism
37 Rate and Pattern of Metabolism in the Blood 109
38 Comparison of HPLC and tic Data 110
39 Comparison of HPLC and tic Data Ill
40 Rate and Pattern of Metabolism in the Blood (HPLC) 112
41 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes ± S.E 113
42 % Distribution Pattern of BaP + Metabolite in Each Tissue 114
Influence of S0? and CAP Administered to IPL on BaP Metabolism
43 Rate and Pattern of Metabolism in the Blood 121
44 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes - S.E 122
45 % Distribution Pattern of BaP and Metabolite in Each Tissue .... 123
46 Rate and Pattern of Metabolism in the Blood (HPLC) 128
Effects of SOp and Particulate on BaP Metabolism
47 % of Total BaP and Total Metabolite Remaining in Each Tissue
at 180 Minutes ± S.E 130
48 % Distribution Pattern of BaP and Metabolite in Each Tissue .... 131
xi n
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TABLES (continued)
Number Page
Biochemical and Physiological Changes in the Lung
49 Biochemical Changes in the Plasma from Blood Perfusing
the Isolated Lung 138
50 Physiological Values in the Isolated Perfused Lung Preparation . . 139
51 Distribution of Metabolites in Lung 144
52 Distribution of Radioactivity 145
53 Perturbations Prior to Perfusion - Concurrent Administration
of Multiple Agents to I PL 146
xiv
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ABBREVIATIONS AND SYMBOLS
BaP -- Benzo(a)pyrene
CAP -- Crude Air Particulate
HPLC — High Performance Liquid Chromatography
ID -- Inside Diameter
IP — Intraperitioneal Administration
IPL -- Isolated Perfused Lung
IT -- Intratracheal Administration
IU -- Internationa] Units
SMC -- 3-Methyl Cholanthrene
OD -- Outside Diameter
ODS -- Octadecylsilane
PAH -- Polycyclic Aromatic Hydrocarbon
Pheno. -- Phenobarbitol (PB)
POPOP -- P-bis(2-(5-diphenyloxazole)benzene)
PPO -- 2,5-diphenyloxazole
tic -- Thin Layer Chromatography
xv
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ACKNOWLEDGMENT
The authors wish to express their gratitude to Carol Warren, Connie
Bools, Janet Dickman, Connie Menefee, Bernadette Nagel, and D. Gary Hancock
for their expert work; Dave Yeager for metal and size analysis of CAP;
Dr. Martha Radike and William Barkley for helpful discussions; and Diane
Dotson for typing the manuscript.
XVT
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A. INTRODUCTION
In the United States lung cancer represents the highest single cause of
cancer deaths (41,58). Thus, there is an imperative need for extensive
studies of causative agents, conditioning factors, and pathogenic mechanisms
responsible for the development of this type of cancer. Animal models have
not been developed sufficiently for the experimental study of this disease.
Although many findings of carcinogenesis studies in other organs and
tissues can be applied to the respiratory tract, a whole range of factors
peculiar to the functional, morphological and biochemical characteristics of
the respiratory organs requires a specialized study of carcinogenic mech-
anisms in the lung. Only recently has the potential of the lung to metabol-
ize foreign substances been recognized as a possible factor of importance in
determining the response of the lung to environmental insults (7,14,16,43-45,
50,75).
Inhalation has been the main mode of exposure in man to agents known
to be casually associated with an increased incidence of respiratory cancer
(41). Epidemiological and experimental evidence (8,12,23,30,32,46) indi-
cates that the interplay of multiple environmental factors is responsible for
the induction of lung cancer. Man is exposed to a complex mixture of
potentially hazardous materials, including specific carcinogens and a variety
of agents which may modify the manner in which the lung disposes of inhaled
materials (40). It is well established that the lungs are capable of
binding and metabolizing such agents (2,7,11,36,43,45,47,52,59,65,73,6,31,
66). One such carcinogen is benzo(a)pyrene (BaP) a ubiquitous environmental
pollutant (3) formed during the destructive distillation of coal and in
other processes that involve incomplete combustion of organic materials
(6,31,66). BaP occurs as both a common contaminant of the urban environment
and a constituent of tobacco smoke. In addition, its metabolites exhibit
varying degrees of mutagenicity, carcinogenicity, and toxicity (24,33,70,72).
A major requirement for understanding the mechanism of BaP carcinogen-
esis is a detailed knowledge of the rate and pattern of formation of
metabolites and the factors controlling their formation. Such factors
include particulate matter, which carries a multitude of chemicals including
BaP and a variety of gases may be deposited in various regions of the
respiratory tract; pollutant gases and vapors may reach the deep lung with
each inspiration, and particulate matter may be largely deposited in the
upper and middle regions of the respiratory tract. Only the smaller parti-
cles of a few microns or less in size reach the deep lung. However, ambient
air of both occupational and urban settings contains many such small
particles.
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It has been established experimentally that BaP in combination with
ferric oxide (53) produces tumors of bronchogenic origin with an incidence of
up to 100%. Carbon particles with BaP also produce a high incidence of lung
tumors (57). The particulate effect has been suggested as a means of
providing longer residence times at the target tissue (12,20,21,29,56,61),
but the biochemical effect has not been fully investigated.
It has been shown experimentally that BaP in combination with S02 pro-
duced squamous cell carcinomas in the rat lung (32,67). However, based on
just this one study and the fact that the control group was small, it is
difficult to draw any firm conclusions about the cocarcinogenic effects of
S02. In another study, rats exposed to high levels of S02 did not produce an
increase in aryl hydrocarbon hydroxylase (AHH) activity of rat lung micro-
somes, while exposure to S02 followed by 3-methyl chloanthrene treatment,
caused an inhibition in the AHH activity when compared to appropriate
controls (26). Besides the aforementioned studies, information on the inter-
action between carcinogenic PAH, such as BaP, and pollutant gases, such as
S02, is still generally lacking. Even though it is well recognized that
95-99% of S02 is adsorbed by the upper respiratory tract (1,42), some
unknown small percentage of S02 will inevitably get into the lower respir-
atory tract. Therefore, this study attempts to determine the effects that
S02 and particulates will have on the metabolism of BaP in the lung at
levels of 1-2 ppm S02. This is well below the industrial threshold limit
value (TLV) of 5 ppm and at levels well within exposure possibilities.
Chronic bronchitis and bronchogenic carcinoma are serious problems that
affect the midregions of the respiratory tract. These diseases are
probably the result of deposition of inhaled materials, such as BaP and
particulates and/or S02, and the effects exerted by clearance of debris from
the deep lung on the ciliary mucous escalator. This debris would include
the inhaled particles, BaP, the metabolites of BaP, and cellular breakdown
products arising from cellular injury and normal turnover. Included in these
cells are the pulmonary alveolar macrophages with their engulfed particles,
enzymes, and carcinogens. Of interest also are such pollutants reaching
the lung via the circulatory system.
Pulmonary disposition of these pollutants has potential importance in
the ultimate toxicity of some agents. While it is true that liver does the
bulk of the metabolic work, it may be that the smaller fraction of the
compound metabolized by or bound to the lung is responsible for disease.
Inhaled carcinogens are an obvious case in point. Although the lungs receive
high exposures of many inhaled contaminants, it must be emphasized that the
lungs are perfused by the entire cardiac output with its supply of compounds
adsorbed from the gut and perhaps those chemicals not yet removed by the
liver. Agents adsorbed via the lymphatics also empty into the venous return
perfusing the lungs. Thus it becomes clear that a better definition of the
pulmonary capacity to metabolize and bind chemicals is necessary.
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There is, however, no way to study the pulmonary metabolism of BaP
in vivo because of the metabolic influence of other organs. In vitro tissue
preparations, such as slices and homogenates, are not satisfactory for
studies involving concurrent administration of multiple agents in different
physical forms (51) or in distribution determinations or binding of compounds
throughout the pulmonary system. Therefore, the isolated perfused lung (IPL)
appears to be the best in vivo preparation for investigating pulmonary
metabolism of foreign compounds (7,43-45), especially compounds adsorbed onto
particulate. A number of criteria were chosen and considered mandatory in
order to provide an isolated perfused lung preparation that was sufficiently
stable to permit evaluation of metabolic activity, distribution, and uptake
of compounds (44), In addition, monitoring of physiological and biological
indices would better define the stability of the system. A summary of the
major features of the isolated perfused rabbit lung preparation is presented
in Table 1.
An important aspect of the current work is the assessment of the rate of
formation and types of metabolites formed when BaP is administered with
CAP or S02 on the IPL. It is well characterized at present, that the meta-
bolic pathway progresses in three directions after possible epoxide
intermediate formation: (a) isomerization and/or hydroxylation, (b) hydra-
tion of epoxides, and (c) conjugation of epoxides (4,18,28,37,48,71,74).
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B. CONCLUSIONS
1. Enzyme Inducers
The data for various inducers on the rate and pattern of metabolism
of BaP are described in Results. In general, it was found that the
metabolites of benzo(a)pyrene formed by this preparation and appearing in
the perfusate resemble in many respects metabolite patterns produced in
the liver. Pretreatment with phenobarbital does not result in an
increased rate of metabolism. Pretreatment with either 3-methylchol-
anthrene or benzo(a)pyrene, however, significantly increases the rate
of metabolism, from 256 ng/hr/g lung to 836 ng/hr/g lung and 1290
ng/hr/g lung respectively. One of the interesting aspects of the
pattern of metabolites formed is the increase in the 9,10 dihydrodiol.
A summary of the conclusions are listed below:
a. PB pretreatment interperioneally causes a decrease in
BaP in the isolated perfused rabbit lung. PB may cause
an increase in an alternate enzymatic pathway or this
increase may simply reflect a relative increase in
oxidation products as reflected by the dione formation.
b. Corn oil significantly increases total metabolism of
BaP in the lung but shunts the metabolism from monohy-
droxylation to 7,8-dihydrodiol formation with activation
of epoxide hydrase system.
c. Both BaP ip and IT pretreatment increase the metabolic
rate of BaP administered IT to the IPL preparation.
A change of the relative percentages of the metabolites,
especially the 9,10-dihydrodiol, is evident in both pre-
treatment groups compared to the control. The BaPTp
pretreatment increased rate versus BaPTT can be accounted
by the corn oil administration.
d. The total metabolic rate of the SMC group can partially
be accounted for by the corn oil administration. Both
IP and IT BaP pretreatment and SMC stimulate 9,10-
dihydrodiol production, whereas the corn oil increases
the 7,8-dihydrodiol and the nonextractable metabolite's.
This interaction may also represent a synergistic effect
of the PAHs with corn oil in the induction of a further
epoxidation step beyond the 7,8-dihydrodiol metabolite
forming the 7,8-dihydrodiol -9,10-epoxide, which has been
indicated as being the ultimate carcinogen. Since this
metabolite is highly reactive, the increase in the 9,10-
dihydrodiol may actually reflect an increase in a further
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hydration product, the 7,8,9,10-tetrahydrotetrol. The
latter product is indistinguishable from the 9,10-
dihydrodiol on our high pressure liquid chromatographic
procedure.
2. Effects of Particulate
The effects of CAP on the metabolism of BaP in the IPL are
described in Results. Work from this laboratory provides evidence
that chemical and physical characteristics of particles may
influence in some manner the induction of tumors besides lengthen-
ing the residence time of the carcinogen benzo(a)pyrene. A
comparison of the rate of formation and type of metabolites
induced after various particle administration may be seen in
Results.
As you will note, CAP pretreatment appears to increase enzyme
activity as indicated by the rate of metabolism. CAP, however
causes a decrease in the rate of metabolism that can be attributed
to an increase in rate of action of macrophages or to a slow
physical release of BaP from particulate. The distribution
indicates that CAP pretreatment decreases the amount of nonextract-
able or polar metabolite and increases the 6,8- and 9,10-diol
formation versus the control. This indicates that the epoxide
hydrase activity causes an increase in the 9,10-diol and an
increase in the diones and monohydroxylated compounds while CAP
with BaP on the IPL following CAP pretreatment causes an increase
in 7,8-diol formation. These data indicate that CAP on the IPL
causes an increase in the epoxide hydrase activity and a slight
decrease in hydroxylation and/or isomerization.
When both CAP and BaP are given together as a pretreatment
followed by BaP on the IPL the rate of metabolism increases to
1093 ng/hr/g lung while the pattern is similar to CAP pretreatment
alone. The rate of metabolism is not an additive effect of the
CAP and BaP (830 and 1290 ng/hr/g lung).
The conclusions reached so far are described below:
1) CAP appears to act through a biological mechanism
such that CAP has a cocarcinogenic effect with benzo-
(a)pyrene. It acts as a physical agent in decreasing
the biological availability or slow release of BaP
over time when administered on the lung with BaP in
comparison with pretreatment of particulate only.
2) The data suggest that CAP affects BaP metabolism by
two different mechanisms: One mechanism appears to
be a long-term effect due to pretreatment with
6
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particulate; this causes an increase in the total
metabolic activity. The other mechanism is a short-
term effect of participate administered to the IPL;
this pretreatment causes a decrease in total metabolic
activity and inhibits the effects of pretreatment.
3) This work helps to partially clarify the ideas of a
number of investigators: a) Particulates used in
maintaining the environment of BaP in the lung for
long periods appear responsible for increased tumor-
igenic response due to the slow release of BaP from
particulate, and b) Particulates appear to influence
metabolic pathways. The indication is that both
factors may be responsible, i.e. slower release of
BaP from particulate as measured by appearance of
metabolites in blood by IPL and a significant change
in metabolic pathway.
4) With concomitant administration of particulate and BaP
and, therefore, a slower release rate, BaP is effec-
tively being administered to the lung tissue in small
doses when compared with BaP by itself. This type of
treatment with particulate appears to be similar to
previous observations in which a carcinogen is much
more effective in producing tumorgenic response when
given in small divided doses over a period of time as
opposed to a single large equivalent dose. In addition,
it is evident that there are differences in the meta-
bolic pathways that seem to be affecting the formation
of the diol expoxide, presently considered by many to
by the ultimate carcinogen.
5) We are presently analyzing the kinetics and binding of
BaP and its metabolites in the IPL to further assess
the significance of these conclusions.
6) The results obtained are very similar whether CAP or
Fe203 is used. Fe203 was used on the IPL under a
grant from NCI, CA-1534403.
Effects of S02
Our attempt is to simulate environmental conditions. The
312 yg of BaP, 1 mg/kg of CAP and 1-2 ppm of S02 used in the
experiments are realistic human exposure values; however, there
are some limitations in our system. The short time period will
show only an immediate effect; with S02 , CAP and BaP together,
it may be necessary to run the experiment for longer periods of
time, perhaps at higher concentrations of S02, or to use larger
-------
amounts of BaP and CAP to obtain dose-response relationships,
or to pretreat with S02 by inhalation. In the presence of
CAP, the S02 can be adsorbed on participates and some of the
S02 may be catalytically converted to bisulfates under the
right conditions of temperature and humidity: in the presence
of sunlight, the S02 can be converted photochemically. We
are more interested at this point, however, in the effects of
S02 on BaP metabolism under our stricter environmental conditions,
A summary of conclusions are listed below:
1) S02 increases the metabolism of BaP by the IPL, and
affects the metabolic pattern slightly. It acts
as a biological agent which causes biochemical
changes in the lung due to irritation. More work
in this area needs to be done in order to answer
this question. The data indicate, however, that S02
can produce changes in the rate of metabolism of a
well-defined carcinogen.
The presence of S02 at 1-2 ppm with CAP and BaP
together indicates that S02 is either adsorbed by
the particulate, or that the BaP is not available
for metabolism due either to BaP not being leached
readily from the particulate, or to an increase in
phagocytic action of the macrophage which may serve
to decrease the amount of BaP available for metabolism.
The particulates, it should be mentioned, can maintain
the environment of the BaP in the lung for long
periods of time, which may be responsible for
increased tumorigenic responses and altered metabolic
pathways.
These studies are a first attempt to determine what
type of interactions take place when S02 and/or
CAP are added with BaP to a lung model system. This
system appears to be a good model system for studying
the metabolism of BaP. The kinetics, distribution
and binding of BaP in the IPL will be analyzed
to determine the importance of these initial studies.
-------
C. RECOMMENDATIONS
It has been demonstrated in this laboratory that particulates,
such as CAP, S02 and Fe203, affects the metabolism of BaP in the
lung. Based on this information the following recommendations are
presented:
1. Information on the dose, size and number of doses of BaP
and/or particulate, particle size and detection of mutagens,
i.e. possible ultimate carcinogens of BaP that are produced
by the lung is needed in order to understand: a) the metab-
olism of BaP under a variety of conditions in the lung and
b) the possible role that pulmonary alveolar macrophage and
the lung tissue play in the mechanism of action of polycyclic
aromatic hydrocarbons. This information can be used in
clarifying the observed pulmonary carcinogenic effects
produced in animal model systems exposed to mixtures of
benzo(a)pyrene and particulate.
2. More information is needed on the kinetics of BaP metabolism,
the binding of the metabolites to BaP, the structure of the
S metabolite, and the better characterization of the non-
extractable materials.
3. More information is needed on the effect of various
particulates or BaP metabolism, distribution and kinetics
in the IPL. The types of particulates that can be studied
are as follows: Fe203, NiO, MnO, A10 and/or diesel fuel
emissions. Additionally, the morphological and functional
changes induced in the macrophages by BaP adsorbed onto
the various particulates need to be determined.
4. There is a need to study the metabolism of BaP adsorbed
on particulate in the whole animal. The material can be
administered IT to the animal with an indwelling cannula.
Metabolism could be studied over a period of days. This
work is important in order to compare metabolism in the
whole animal with that observed in the IPL.
5. At the same time metabolism studies are carried out
(part 3 and 4), whole animal carcinogeneous studies need
to be carried out in order to determine the biological
responses induced by BaP adsorbed on particulate. It will
be important to look at the effects of these materials
given IT to the whole animal with and without pretreatment.
We have found that pretreatment with and without particulate
enhances the metabolic rate and changes the metabolic pattern,
The underlying question is "Does an increase in metabolism
shorten the latency for a biological response?"
-------
6. Morphological and functional changes induced in pulmonary
alveolar macrophage (RAM) by BaP adsorbed on particulate
in long-term studies such as those described in part 5
need to be carried out. Changes in RAM can be monitored
over time. These studies would involve looking for effects
that may or may not be related to biological responses.
7. The IPL should be developed for use as a screening tool
for the determination of potentially cocarcinogenic agents.
10
-------
D. MATERIALS & METHODS
1. Chemicals
ik
(7,10- C)-BaP, 21 mCi/mmole, is obtained from Amersham Chemical,
Arlington Heights, Illinois, while unlabeled BaP is obtained from
Aldrich Chemical Company, Milwaukee, Wisconsin, and 3-methyl chloan-
threne from Sigma, St. Louis, Missouri. These compounds are checked
for purity by tic and HPLC. If necessary, the cold BaP can be
purified further by the use of neutral alumina column chromatography
with benzene or toluene as the eluant followed by recrystallization
in a benzene-isopropanol mixture.
Dr. Harry Gelboin of NCI supplied BaP metabolite standards,
3-hydroxy, 3,6-,6,12-, and 1,6-quinones, 9,10-dihydrodiol (9,10-diol),
7,8-dihydrodiol (7,8-diol), 9-hydroxy, 7-hydroxy; Dr. Ronald Harvey
of Ben May Cancer Institute supplied the cis and trans 4,5-dihydrodiol
(4,5-diol), 4,5-epoxide, and 4,5-quinone; and Drs. Tom Meehan,
Ken Straub, and Joe Landolph of the Chemical Biodynamics Lab.,
University of California, Berkeley, provided the (+) 7a,83-dihydroxy-
93,10B-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (7,8 diol-9,10-
epoxide), and 6- and 7-hydroxy of BaP. The crude air particulate
(supplied by EPA) from the Pittsburgh vicinity, analyzed by the
analytical division of Kettering Laboratory for size and metal content,
contains particulate size 79.0% < 10 Mm, 64.5% < 6 um, 47.5% < 4 ym,
and metals (ppm): Cd, 40; Cr, 542; Fe, 47,976; Mg, 6,482; Mn, 2,219;
Ni, 1,417; Pb, 6,818; Zn, 5,496. The CAP has been partially character-
ized for polynuclear aromatic hydrocarbon (PAH) content. One gram of
CAP is extracted with 15 ml chloroform after mixing for 3 hr; the
chloroform extract is then concentrated and analyzed by HPLC with
UV detector. The effluent is collected and further analyzed by
fluorescence. The following compounds have been identified by
flourescence and known standards: carbazole (43.7 ppb), fluoranthene
(77.9 ppb), pyrene (66.8 ppb), benzo(k)fluoranthene (141 ppb),
benzo(a)pyrene (32.8 ppb), benzo(ghijperylene (47.8 ppb). Benzo(c)-
phenanthrene and phenanthrene have been tentatively identified
(Table 2, Fig. 1).
For scintillation counting, PPO (2,5-diphenyloxazole) and
Scintiverse media & POPOF Qp-bis-(2-(5-phenloxazol benzene)} are
obtained from Fisher Chemical, Cincinnati, Ohio, while Triton X100
is obtained from Rohm and Haas, Cincinnati, Ohio and corn oil is
obtained, from Mazola Best Foods, Englewood Cliffs, N.J.
Heprin is purchased from Abbott Laboratories (North Chicago, 111.),
sodium phenobarbital, lidocaine 2% and epinephrine from Parke Davis
(Detroit, Michigan). Pararosaniline is obtained from Eastman Kodak
(Rochester, N.Y.) and ninhydrin spray from Sigma (St. Louis, Mo.).
11
-------
All solvents used are redistilled except methanol (Fisher Scientific,
Fairlawn, N.J., spectroanalyzed and HPLC grade).
2. Instrumentation
All 14C scintillation counting is performed on Tri-Carb Packard
Liquid Scintillation Spectrometer (3200 and 2002) and fluorescence is
recorded on an American Instrument Corrected Unit. IPL samples are
chromatographed on a Varian 8500 HPLC with variable vv wavelength
detector under the following conditions: Varian reverse phase ODS
25 cm x 2 mm columns, 10 ym particle size, methanol-water mixture from
62% to 100% methanol; increasing 1%/min x 6, 0% for 3 min, 3%/min x 3;
3%/min x 6 and 4%/min x 6; room temperature; monitor at 268 nm; flow;
1 ml/min.
3. Isolated Perfused Lung
a. Perfusion Apparatus
The system for perfusing the lung (Figure 2,3) consists of three
integrated components: 1) the ventilating gas; 2) perfusing apparatus;
and 3) an artificial thorax. The portion of the system in direct con-
tact with the blood is glass with the exception of silicone rubber
tubing (Size AF - New Brunswick Scientific, International) used in the
peristaltic pump. The glass is treated with Si lie!ad according to
directions of the manufacturer (Clay Adams).
1) Ventilating Gas
Filtered air is provided by a diaphragm type air pump (Figure 2,3).
The rate of flow of carbon dioxide and air are measured with rotameters
and the rate regulated by adjustable clamps. The gases are mixed and
their entry into the system is regulated by a solenoid valve (A). This
solenoid, as well as an additional one in parallel (B), are activated
by the movement of a rubber breathing bag against a double-pole micro-
switch. The solenoids operate in unison, one opened and one closed
(see Figure 5). This provides a stream of gas at a constant pressure
(0.25 cm H 0) to the lung.
The ventilating gas is warmed before it enters the lung by passing
it through a glass coil submerged in a waterbath, maintained at 37°C,
and is then humidified by bubbling through a coarse glass frit washing
bottle. Excess water is removed by a trap. After passing through a
rubber breathing bag, undirectional flow is maintained by a one-way
valve, and the pressure is monitored by a water manometer. A three-way
teflon, 4 mm bore, stopcock (Gj) permits the addition of other gases,
aerosols, etc. to the ventilating gas.
12
-------
FIGURE 1
Composition of Crude Air Participate
Fe
Cd
Ni
Zn
Cr
Pb
Mn
Cu
JllJlllllllllllIJllllllJUlUlllllilUl|lIUUlliUllll|l^
illMIIIIIIIIIMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIHIIIMIIIIIIl
Crude Air Particulate Illllll
Coke Oven ParticuloteBBB
Coal Dust "x^*-"*
Cool Tor
imnmim
innii|||||||iiMuiiMii
iiniiiiiiiiiiimimiiniM
0,1
i.o
10 10* 10
Metal Concentration 0-jg/g)
i4
i5
-------
TABLE 2.
EMISSION SPECTROGRAPHIC ANALYSES OF CRUDE (AIR)
PARTICULATE MATTER
Metal
Aluminum
Antimony
Barium
Beryl 1 ium
Bismuth
Boron
Calcium
Cobalt
Copper
Molybdenum
Phosphorus
Potassium
Sil icon
Silver
Strontium
Tellurium
Thai 1 ium
Tin
Vanadium
Concentration (ppm)*
10,000
0
1,000
0
0
500
10,000
10
1,000
1,000
5,000
0
10,000
0
500
0
0
500
500
*These sensitivities may be too low for the more volatile metals such as
antimony, thallium and tellurium.
14
-------
A fork-shaped tube with one-way valves joins the ventilating gas
system through a tee-connection that permits the lung to respire fresh
ventilating gas with each inspiration. Extratracheal dead air space
is 2.5 cc. The expired gas may be diverted to a spirometer or to an
exit gas line through a three-way teflon, 4 mm bore, stopcock (G2).
(See Modification Section e)
2) Blood Perfusion System
A water jacketed blood reservoir (manufactured by Thomas Curcar,
792 Kenray Court, Cincinnati, Ohio) (50 ml) provides a constant hydro-
static pressure (23 cm H20 measured at the hilus) to the cannulated
pulmonary artery (Figure 4). The level of blood is maintained in the
reservoir by a sensing device (Dyna Sense electronic liquid level con-
troller - Cole Farmer) using platinum-tipped electrodes which control an
off-on switch to a peristaltic pump (Harvard Apparatus Model 1210).
The teflon, 2 mm bore, stopcocks, (Rx and R2) are used only in the
start-up procedure to ensure air-free lines. Blood drains freely from
pulmonary vasculature and is exposed to the cycled sub-atmospheric
pressure in the artificial thorax. The bottom portion of the artificial
thorax serves as a reservoir for the venous blood.
The pH is monitored continuously by placing the electrode directly
into the 50 ml reservoir through a port in the plexiglass top.
An infusion pump (Harvard Apparatus - Model 1131) delivers the
additives, i.e., glucose and dosium bicarbonate, at a constant rate
into a port of the 50 ml blood reservoir.
3) Artificial Thorax
A temperature of 37°C is maintained in the entire system by
circulating the water from the constant temperature bath (37°C) through
the jacketed blood reservoir and artificial thorax by means of a sub-
mersible water pump (Figure 2 - Sargent Welch Sci. Co. S-7151C).
A plexiglass lid, fitted with a rubber "0" ring, and scaled with sili-
cone high vacuum grease is held in place on the ground glass rim of
the thorax.
Three ports protrude from the lid. One port is connected to the
small animal respirator (Harvard Apparatus - Model 662) which is
operated in reverse. Another port provides the controlled vacuum source
and the third port leads to a Magnehelic gauge (Dwyer Instruments Inc.)
to monitor thoracic pressure.
Four glass prongs, which extend horizontally from inside the arti-
ficial thorax, provide a rest upon which a circular piece of tygon
tubing rests. Gauze strips saturated with distilled H20 are hung from
the tubing and provide a humidified interior.
15
-------
I PL Design
Level Sensors
Infusion Pump
Constant Pressure
Blood Reservoir
To Manometer
Microswitch _ Waste Shunt
Gas Exit
Spirometer
Forked - shaped Tube
y Needle Valve
acuum
Respirator
Gauge = Rota- »
Warming
Coil
«metersE
Artificial Thorax
Humidifying Shroud
Humidifier
Water
Pump
Peristaltic
Pump
-------
pH Meter
Level
Sensor
Respirator
a
Vocumn
Peristaltic
Pump
FIGURE 3
Simplified I PL Schematic
Constant
Pressure
Reservoir
Artificial
Thorax
Infusion
Pump
Carbon Dioxide
'Respiratory
Valve
Complex
Warming
8
Humidifying
Solenoid
Complex
Filtered
Air
-------
FIGURE 4
Cannulae for IPL
5 cm.
1 I
Pulmonary
Arterial
Tracheal
Left
Afrial
18
-------
FIGURE 5
Solenoid Diagram
Air
Solenoid A £
To Lung
Warming
otc.
Double-polo Microswitch
-------
b. Preparation of Lungs
The rabbit is tied on a specially designed slanted board with the
head below the elevation of hind legs and the hair removed with electric
clippers from the ventral side. Heparin (1000 lU/kg body weight) is
injected intravenously into the median vein or central artery of the
ear. Five minutes a cardiac puncture is made using disposable 60 ml
syringes (18 gauge needle). At least 85 ml of blood is needed to
prime all lines, and to sample periodically from the system. (Our
experience has enabled us to figure on recovering at least 21 ml of
blood per kg body weight.) Care is taken to enter between the sixth
and seventh ribs next to the sternum so as not to puncture the lungs.
Immediately following the cardiac puncture the rabbit is killed by a
sharp blow to the head or C02 inhalation. A midline incision is made
from the neck to the abdomen to expose the trachea and rib cage. The
liver is retracted and the diaphragm cut on both sides to collapse the
lungs. The heart and lungs are exposed through a midline sternotomy and
the rib cage retracted. Additional blood may be recovered at this point
if an adequate amount was not obtained via cardiac puncture.
The trachea is then cannulated using a siliconized (Siliclad-Clay
Adams) glass tube (3 mm ID, 4 mm OD by 3.5 cm in length) and ligated
(see Figure 4). The trachea, lungs and heart are dissected free from
their attachments, taking care not to puncture the lungs, and kept
moist with physiological saline.
The pericardium is removed and the pulmonary artery cannulated with
a siliconized glass tube (3 mm ID, 4 mm OD, by 6.5 cm in length) pre-
viously filled with heparinized blood and ligated. During this proce-
dure care must be taken not to introduce air bubbles into the vascula-
ture or an immediate cessation of flow occurs. The entire right
ventricle and right atrium, together with most of the left ventricle
(up to 0.5 cm below the A-V septum) is removed. The left atrium is
cannulated by passing a siliconized glass tube (3 mm ID, 4 mm OD, by
7 cm in length) through the remaining left ventricle and bicuspid valves
to the atrium. The cannula is secured with a ligature and the remaining
tissue dissected free. The preparation is weighed.
c. Starting the System
One hour prior to perfusion, the water bath and pump are switched
on so that the temperature of the system reaches 37°C. All tubing that
carries blood, the blood reservoir, and the artificial thorax are
flushed with physiological saline and drained through a 3-way teflon,
2 mm bore, stopcock (D). The arterial tube is filled with heparinized
blood up to the ball and socket joint, a 12/5 fitting (S), and the
teflon stopcock (R2) closed. The reservoir is filled to the proper
level taking care to avoid bubbles of air from becoming trapped in the
lines and then the teflon stopcock (Rj is closed.
20
-------
The lung preparation is suspended in the artificial thorax by
connecting the tracheal and pulmonary arterial cannulae, using silicone
rubber tubing, to the appropriate glass tubes. The tubes pass from
the thorax through a No. 8 rubber stopper mounted in the plexiglass lid.
At this point the 50 ml reservoir is brought into position and the
ball and socket joint clamped. Stopcocks RI and R2 are opened to allow
the blood to flow freely. All blood previously collected is slowly
added to the reservoir and the peristaltic pump turned on, intermittent-
ly, to fill the tubing. As soon as all tubes fill, the remaining blood
is added, followed by heparin, and epinephrine. The constant level
probes, extending through a plexiglass top, are placed into the
reservoir to ensure a constant blood pressure of 23 cm of blood measured
from the hilus of the lung, and the peristaltic pump is activated. It
is emphasized that extreme care must be taken so as not to introduce
air bubbles into tubing carrying blood between the blood reservoir and
the lung via the pulmonary artery.
The remaining connections on the lid are completed. The air
pump, C02 cylinder, rotameters, and solenoid valves are activated or
adjusted (air flow - 3.3 L/min., C02 5%) allowing the prewarmed and
humidified gas mixture to flow.
The vacuum source is then turned on and adjusted by a needle
valve to reduce the pressure inside the artificial thorax initially to
-25 to -20 cm H20, which opens the collapsed lungs. The respirator is
started and alternating sub-atmospheric pressures (-3 to -12 cm H20) are
maintained by adjusting the stroke volume of the respirator and the
needle valve. The frequency of breathing is kept at 50 respirations per
minute.
At this point, the system is completely automated except for
occasional large sub-atmospheric pressure excursions (-30 cm H20) every
15 minutes, to sigh the lungs by closing the needle valve.
Glucose (30 mg/hr) and sodium bicarbonate (0.3 mEq/hr) are added to
the reservoir with a constant infusion pump at a rate of 0.3 ml/hr.
The pH (7.35 to 7.45) is maintained by adjusting the quantity of C02
mixed into the air stream. Additional heparin and epinephrine are
added as needed to the blood through the blood reservoir or administered
through the forked-shaped tracheal tube if fluid, or if gas or aerosol,
by the teflon stopcock (Gj). Blood samples may be drawn from either
stopcock (D) or the reservoir at various intervals of time throughout
the perfusion.
The rate of blood flow was estimated by allowing continuous flow
through the peristaltic pump, calibrated at 37°C and measuring the vol-
ume of blood collected per unit of time. Net lung weight gain was used
as one indicator of edema formation.
21
-------
d. Cleaning the System
Immediately after an experiment, the system is flushed three times
with distilled water. The system is then partially filled with a 10%
solution of Isoterge (Scientific Products), and allowed to circulate
for 15 minutes. This is then followed by five additional rinses with
distilled water. The cannulae are soaked in an Isoterge solution for
2-3 hours and rinsed thoroughly with distilled water. The silicone
rubber tubing is changed after each perfusion.
e. Modification
One of the aims of this study has been to study BaP coadministered
intratracheally with CAP or SO . A development which arose from this
need was the tracheal valve system which is shown in Figure 6. The
valve is fabricated with Teflon and has an extra tracheal dead air
space of approximately 6 CM . Silicone rubber stem valves permit
unidirectional flow. The offset diagram in Figure 6 gives the dimen-
sions of the valve extension mold which is also fabricated with Teflon.
Intratracheal pressures can be measured and intratracheal instillations
are made through a point at the top of the valve. Spirometric measure-
ments are also possible, therefore adding another diversion to metabolic
and acute toxicity investigations. This modification replaces 62 and
the spirometer indicated in Figure 2 and the tracheal valve is
connected to the fork-shaped tube.
22
-------
FIGURE 6
Trachea! Value Schematic
ro
CO
SILICONE RUBBER GASKET
d c
I i-|
J:
dia
p '
-;L>
i i
>!ir
LJ!
c-H
q
/
g
\L
•4 C
\
\
1
1
1
If"
_-•"
_*.
*|
1
1
1
K
SILICONE
RUBBER
VALVE
EXTRUSION
MOLD |
dia
dia
c
la
Tt
c d
21 Gage S.S.Tube
dia
dia
-------
f. Pretreatment of BaP, CAP, SMC, Phenobarbital and Corn Oil
IP pretreatment with BaP or SMC, 20 mg/kg in corn oil vehicle is
performed 24 hours before sacrifice, sodium phenobarbital 50 mg/kg in
saline on three successive days with last dose 24 hours before sacrifice
and corn oil, 3 ml/kg, 24 hours before sacrifice. Intratracheal in-
jection of 10 mg/kg of CAP and/or BaP based on work by Saffiotti (53)
with hamsters is carried out once a week for 5 weeks. The particulate
is suspended in 2 ml of physiological saline. The animal is restrained
on the rabbit board, with the level of the head slightly below the level
of the heart, the neck region shaved with small animal clippers, and
then palpated to locate the trachea. A 16-gauge, 1.5 inch needle with a
12-ml syringe attached is inserted through skin into the trachea. If
8 to 10 ml of air can be removed without resistance, the needle is in
the trachea. Following injection, the needle is removed, the head of
the rabbit raised (rabbit board tilted to 45° angle), and the animal
is forced to breathe deeply by applying periodic pressure to diaphragm.
g. Administration of BaP, CAP to I PL
One microcurie of pure (7,10-14C)-BaP (21 mCi/mmole, Amersham/
Searle, Arlington Heights, 111.) is diluted with unlabeled pure BaP
(Aldrich Chemical Company, Milwaukee, Wis.) and evaporated gently to
dryness under nitrogen. It is then taken up to a final amount of 1.24
ymoles of BaP (0.8 pCi/mmole) or 312 yg of BaP in a 1-ml ethanolic
saline (1:1) solution and is intratracheally injected on the IPL. When
BaP and particulate are added together on the IPL, the solutions of
labeled and unlabeled BaP are slowly evaporated under a stream of
nitrogen so that the BaP is adsorbed onto particulate and then taken up
in 1 ml of saline and intratracheally injected on the IPL. In each case
the syringe is rinsed once with an additional milliliter of saline and
injected on the IPL.
24
-------
h. S02 Administration In Vivo and In Vitro
For the generation of S02, a stream of dry compressed air is passed
over a 2 inch 3/16 in. i.d. x 0.30 in. wall thickness FEP teflon per-
meation tube with FEP teflon plugs maintained at 37°C containing
condensed S02 (54). This S02-air stream is then mixed with either
humidified air containing 5% C02 from the ventilating system of the IPL
before entering the IPL preparation via the trachea! valve (Fig. 7) or
dry filtered room air before entering the tracheotomized rabbit (Fig.8).
The exhaled and excess S02-air is then bubbled through two sodium
hydroxide scrubbers and a dessicating column before it is vented out of
the system by a house vacuum. The concentration of S02 is adjusted by
carefully controlling the following systems: the stream of compressed
air over the permeation tube; the dry room air (in vivo) or humidified
C02-air (in vitro) and the vacuum source. A critical resistance con-
sisting of a 3 in. piece of P.E. 60 tubing is used to measure flow rate
of the compressed air. The West Gaeke method, a colorimetric determin-
ation of sulfur dioxide concentration (ppm) is used (27,69). Air is
sampled for specified periods of time throughout the experiment by a
midget impinger containing 10 ml of 0.04 M potassium tetrachloromercur-
ate absorbing reagent located on the exhaust side of the system. Two
ml of a 0.016% pararosaniline (Eastman Kodak Co., Rochester, N.Y.)
reagent and 1 ml of a 0.2% formaldehyde solution are added to the
absorbing reagent, transferred to 25 ml glass stoppered graduated
cylinder, mixed, and the purple color is allowed to develop for 20
minutes at which time the absorbance is determined at 575 nm on a
Beckman yv spectrophotometer. The micro!iters S02 of the samples are
determined from standard calibration curves of absorbance (range 0 to
0.6) versus micro!iters or micrograms of S02. The standard curve is
prepared daily. Concentration (ppm) of S02 is determined by dividing
microliters S02 by the flow (liters/min) that the vacuum draws through
the impinger. The entire system is equilibrated with the S02 in line
to obtain 1.5-2 ppm before placing the rabbit or the lung in line and
the S02 is then monitored for the duration of the experiment. The
SO pretreatment is performed by restraining the animal on the rabbit
board with the level of the head slightly lower than the level of the
heart and the neck regions shaved with a small animal clipper. One cc
of local anesthetic, lidocain 2% in an epinephrine solution (W. A.
Butler Co., Columbus, Ohio) is administered subcutaneously to several
sites, injecting a total of 3 to 4 cc. After 5 minutes, a 1 inch mid-
line incision is performed, the trachea isolated, a small horizontal
cut is made in the trachea, and the cannula inserted and ligated. A
tee connection in line with the smallest possible distance to the
cannula is attached to the cannula through which the rabbit breathes.
An additional air reservoir is added to aid the rabbit in case of a
sudden loss of positive pressure. A slight positive pressure is main-
tained by monitoring a respiratory bag placed in line immediately after
the rabbit and before the air trap. A rotameter placed on the inhala-
25
-------
tion side of the rabbit is used to monitor the rabbit's respiration.
In these experiments the average respirations prior to tracheotomy are
167 respirations/minute and after tracheotomy 153 respirations/minute
while the pulse rate remains unchanged at 80 pulses/minute.
26
-------
FIGURE 7
S02 Modification of IPL
IN VITRO
ro
dessicatmg
column
air
microliter
flow valve
^pressure
and flow gauges
humidified
air
water bath
( 37°C)
perfused
lung
rota meter
permeation
tube
mixing
chamber
NaOH
scrubber
vacuum
0
dessicating
column
-------
FIGURE 8
Modification of IPL
IN VIVO
r\3
oo
dessicating
column
air
m
I
;>-^
Sy
room air
pump
C
micrometer
flow valve
i one-way — ^
' valve
i 1
i T—>
6X v
pressure
and flow gauges ^
water bath
( 37°C )
s
\
m
permea
tube
~~~^ ~- rota meter
r .s
/ midget
/ impinger
/ sampler
7 P| 1 M I 1
L-U \ [I "J
k rabbit |
) trap
.V mixing LJ JJ L
chamber
tion Na°H
scrubt
vacuum
- — o
trap 1— m
5:|
n ^
( ^ dessicating
\ / column
)er
-------
i. Extraction and Analysis of Biological Samples
5.5 ml blood samples are taken from the perfusion system at
15,30,60,90,120, and 180 minute intervals. Of this volume, 0.5 ml is
placed in a glass scintillation vial, digested with 0.5 ml IN NaOH in
a 60°C oven overnight, bleached with 0.5 ml tert-butyl hydroperoxide in
a 60°C oven for thirty minutes, and then counted with 15 ml of
Scintiverse (Fisher Scientific) Scintillation Media with internal
standard. This sample is the pre-sample, i.e. prior to extraction.
The remaining 5 ml of blood for each sample is extracted twice with
30 ml acetone, benzene, isoamyl alcohol (10:13:0.1) by shaking for 30
minutes and then centrifuging at 1500 rpm, 5°C, for 30 minutes. The
organic phase is removed and evaporated to dryness in a 50°C water
bath, under nitrogen and stored in the cold until analysis. The
remaining tissue residue (post extraction) is digested in 10 ml IN
NaOH overnight in a 60°C oven and a 0.5 ml sample is bleached and
counted as above. The organic portion is reconstituted in benzene
(O.^J6 ml) for thin layer chromatography (TLC) or in chloroform (0.15 ml)
for high pressure liquid chromatography (HPLC).
When the lungs have been removed from the perfusion system they
are weighed (to determine weight gain due to edema) and then lavaged
three times with physiological saline (5 ml/g lung tissue). This
"washout" fluid is then centrifuged at 1500 rpm, 5°C for 50 minutes in
250 ml centrifuge bottles. The supernatant is decanted in a 500 ml
graduated cylinder. The pellet, containing pulmonary alveolar macro-
phages and any possible particulate remaining from injection, is
transferred to a graduated centrifuge tube along with approximately
20 ml of "washout" fluid and recentrifuged. The supernatant fractions
are combined in the 500 ml graduated cylinder. This volume is recorded,
and 1/3 of this volume is extracted twice in a 250 ml separatory funnel
with an equal volume of extraction solvent. A pre-washout 0.5 ml
sample is digested in 0.5 ml IN NaOH in a 60°C oven overnight, bleached
and counted as above samples. Following the second extraction, the
tissue residue volume is measured, 0.5 ml (post sample) is digested
with 0.5 ml IN NaOH in a 60°C oven overnight, bleached, and counted.
The organic portion of the washout is evaporated in a 50°C water bath,
under nitrogen and stored in cold until further analysis. The organic
portion is reconstituted in benzene •t&.jfifi-flrl-) for TLC or (.15 ml)
chloroform for HPLC.
The macrophage pellet from above is resuspended in distilled
water to a total volume of 5.0 ml, 0.5 ml is digested, bleached, and
counted and the remaining 4.5 ml is extracted twice with 30 ml of
extraction solvent, and centrifuged. The organic portion is evaporated
under nitrogen in a water bath as in above sample and stored__in colcU.-— -
until analysis. The organic portion is reconstituted i nQK 6 jn) benzene
for TLC and .15 ml chloroform for HPLC. The tissue residue is digested
with 10 ml of IN NaOH overnight in a 60°C oven, volume recorded, and
29
-------
0.5 ml bleached and counted as described above.
After the lungs have been lavaged, all extraneous tissue is
removed (including left atrium of heart, fat, etc.) then the lung
tissue is scraped from the trachea bronchi. This is done by scraping
the tissue with a scalpel. After as much lung tissue has been removed
as possible, the weight of that tissue, as well as the weight of the
trachea-bronchi is determined. The lung tissue is homogenized in a
Waring Blender (2 minutes) with sufficient distilled water to make a
total volume of 75 ml, and a 0.5 ml sample is digested, bleached and
counted. After homogenization of the lung tissue, 10 ml is placed in
a 50 ml graduated centrifuge tube and extracted twice with extraction
solvent. After the second extraction, the organic portion is evaporated
under nitrogen in a water bath and stored in cold until analysis. The
organic portion is reconstituted in 0.6 ml benzene for TLC or .15 ml
chloroform for HPLC. The tissue residue is digested with 10 ml of
IN NaPH in an oven overnight, post volume recorded, and a 0.5 ml
sample bleached and counted.
The trachea bronchi are cut in small pieces, placed in a 50 ml
graduated centrifuge tube, and extracted twice with acetone, benzene,
and isoamyl alcohol. No presample is able to be taken from the trachea
bronchi. The organic portion is evaporated^under nitrogen, stored in
A cold until analysis, and reconstituted in d^m1 benzene for TLC and
'^>^- .15 ml chloroform for HPLC. The tissue residue is digested with 10 ml
of IN NaOH overnight in a 60 C oven. The post volume is recorded, and
a 0.5 ml sample is bleached and counted.
Metabolite standards are dissolved in benzene or methanol for TLC
and in methanol for HPLC. A portion of the reconstituted benzene sample
is chromatographed on TLC (0.1 mm silica gel plates, Eastman Kodak 6061)
using benzene-ethanol (19:1). Spots are located in the yv light, the
entire chromatogram is cut into strips, and the spots are quantitated
by counting using a cocktail of POPOP and 4 g PPO per liter of toluene.
Identification is made chromatographing metabolite standards with each
experimental sample.
An HPLC chromatogram is recorded using the standard mixture
(Fig. 9,10) once each morning. The chloroform sample is then chroma-
tographed and fractions collected with time, such that each of the peaks
and spaces between peaks are collected individually. The fractions are
then quantitated by counting using a cocktail of toluene:Triton X100:
ethanol (8:4:3) plus PPO (6 g/liter) and POPOP (25 g/liter). For both
TLC and HPLC, amounts of each metabolite are determined for each sample
in nanograms per gram lung wet weight used for each perfusion. The
total rate of appearance of metabolites (ng/hr/g lung) in the blood is
based on a linear regression of a time count study from 0 through at
least 90 minutes. The metabolites in the washout, lung, trachea
bronchi, and macrophage are also determined in nanograms per gram lung
30
-------
wet weight. The trachea bronchi is initially determined based on
trachea weight and then is corrected to lung wet weight. All data
collection, data reduction, and statistics are handled by computer
programs. All samples are processed under nitrogen and subdued
yellow lighting to minimize photo-oxidation.
This work was begun using TLC methods before HPLC methods were
routinely used in the laboratory. Therefore, the HPLC data presented
validate the TLC data as indicated in Results. The TLC blood data are
presented as the slope over the range of 0-120 minutes and as distri-
bution data at 180 minutes. The HPLC blood data are compared to the
TLC blood data either at 60 minutes only or using the slope over the
range of 0-120 minutes.
The data for the 9,10- and 4,5-diols are directly comparable
between the two techniques. The monohydroxylated metabolites on TLC
are composed of 14C-activity under the 9- and 6-OH and 3- and 7-OH
peaks on HPLC. Dione metabolites on TLC are composed of 14C-activity
under the 4,5-quinone, 3,6-,6,12- and 1,6-quinones and 4,5-epoxide
(or its derivative) peaks on HPLC. The 7,8-diol and the S metabolite
on HPLC, chromatograph together on TLC.
Scintillation counting indicates that an unknown (S) metabolite
chromatographs about 1 minute after BaP. When this fraction is col-
lected and chromatographed on TLC in benzene:ethanol (19:1) 14C-actiVity
appears in a spot above baseline which is both fluorescent and ninhydrin
positive. This suggests that the S metabolite is either an extractable
peptide conjugate or part of the nonextractable metabolite (S) which
chromatographs very differently on HPLC and TLC. The S metabolite
varies with in vivo treatment and this suggests that the metabolite is
an extractable peptide conjugate. Chromatography of these same samples
on TLC results in a ninhydrin positive test for the 7,8-diol spot which
indicates that the S metabolite is part of the 7,8-diol spot. The HPLC
data indicate that the 7,8-diol TLC spot contain approximately 3-4%
7,8-diol. The rest of the percentage in the 7,8-diol spot is attributed
to the S metabolite as derived from HPLC data. The S metabolite is
still under investigation.
The low levels of quinones, in particular the 5,5-quinone, as mea-
sured by HPLC and TLC methods are indicative of little oxidation due to
workup procedures. In addition to retention times corrected fluo-
rescence spectra of concentrated extracts of 50 ml of blood were chroma-
tographed on HPLC and the various metabolite fractions were collected
and their fluorescence spectra were compared with known standards 9,10-,
7,8- and 4,5-diols and the 90 and 3-OH. The quinones and 7-OH were not
identified by this method. The 6-OH and 4,5-epoxide are shown to indi-
cate relative retention times of these metabolites.
31
-------
TABLE 3.
BaP STANDARDS ON tic
Compound
Rf Value
10
B(a)P
765
0.88
3,6-dione
0.78
3-hydroxy
0.48
HO H
H
'OH
4,5-dihydrodiol (P2)
0.24
HO
H OH
7,8-dihydrodiol (Baseline)
0.03
OH
HO
9,10-dihydrodiol (p )
0.16
32
-------
FIGURE 9
HPLC of BaP Standard Control
50r
SOLVENT PROGRAM-GRADIENT SOLUTION
I 2345 f> 789 10
Slep •••••••OOO Abs 0- 5,X=268nm
RotexOOl OOOOOOOOOO Chart 5 in/min
mull. xO.I OOOOOOOOOO
I 9O99OOOQOO Sol A Water
Rote 2 OOOOOOOOOO Sol B Methonol
%b/mm.4 OOOOOOOOOO A»B Flow role 60ml/hr
8 OOOOOOOOOO Initial b ^ 62 Final b=IOO
Decrease OOOOOOOOOO Step interval 3 mins.
25
0
x2 OOOOOOOOOO Temp. 2-3" C
x5 OOOOOOOOOO Somple size 15
/Jl .^
Reset OOOOOOOOOO Somple mix 14 j_,^rJ~'
_rr'J'
-^-J~J~~' 3,6-
9,10 diol , 6.
^ benzo[o
_ CHCI3 4,5 diol 6,'l2-qumone
\
1
Mr
A A 7.8 diol
benzene 1
\ 4'5 A<
\ 1 quinone H^
M , A V0*/
\ / v/ v_ /VJv
i i i
0 5 10 15
9- OH r 7- OH
6-OH
- A A3-OH
' \ *A
d/ \ / 1
pyrene
U
J l l
i
20 25 30
TIME (MIN)
33
-------
FIGURE 10
HPLC Chromatogram using HiBar II Reverse Column
Column - 10 u particle size, 25 cm x 4.6 mm
0.20 r
CO
-p.
BoP
15
TIME (MIN.)
-------
E. RESULTS
1. Effects of Enzyme Inducers
In Table 4 the various pretreatments used are listed. Table 5
shows the influence of enzyme inducers on the total metabolite
appearance in the blood. IP pretreatment with Pheno. does not signifi-
cantly influence the total rate of metabolism of BaP in the I PL. In
fact, there appears to be a decrease in rate when compared to its
control. BaP given IT or IP significantly induces BaP metabolism over
control; BaPjp is approximately 3 times the corn oil control while
BaPjj is approximately 5 times the non-pretreated control. Corn oil
was found to increase the total rate of metabolism about twofold over
the nonpretreated control while SMC was found not to be significantly
different from the corn oil control. It is perhaps interesting to note
that the rate of BaPjj plus the corn oil rate is approximately
equivalent to BaPjp.
a. Influence of BaP Pretreatment
BaP pretreatment IP or IT causes a large increase in the rate of
metabolism of BaP (Table 6). This is due to the fact that BaP will
induce the P^SO (P448) enzyme system (64). The metabolic profile
shows a marked increase in the 9,10-dihydrodiol for BaPjp or IT pre-
treatment while there is a significant decrease in the monohydroxylated
and diones for BaPjj from the control and BaPjp pretreatment. The
nonextractables for the control and BaPjj are comparable while the
value for BaPjp is considerably smaller.
These data suggest that even though BaP is given by two different
routes of administration the rates of metabolism are significantly
higher than the control in both cases and similar enzyme systems are
induced. In one case the enzyme levels are increased in whole animal
but especially in the liver 24 hrs. later, while in the second case
the enzyme levels are increased specifically in the lung over a six
week period. However, the metabolic patterns do show differences in
that larger amounts of phenols and diones are formed, and therefore,
less nonextractable materials for BaPjp pretreatment. The nonextract-
ables may indicate that there is less material available for binding,
i.e. dihydrodiols or epoxides.
HPLC supports the TLC data (Tables 7,8, & 9) (25,55). Four BaP
Control experiments in Table 7, show that the TLC is very comparable
to the HPLC. In addition the 7,8-dihydrodiol and the S metabolite
(which appears after BaP on HPLC) co-chromatograph on TLC and are
comparable. The chemical characterization of the S metabolite is un-
known at present; it is fluorescent and ninhydrin positive.
35
-------
Data for BaPjy and BaPjp pretreatment experiments in Tables 8
and 9 show comparable results for HPLC and TLC. It should be noted,
however, that these are 60 minute time points and not values from a
slope. There are some discrepancies in the diones and phenol values
but that is to be expected considering the differences in techniques
36
-------
TABLE 4.
PRETREATMENT REGIMEN OF ENZYME INDUCERS
Group
Control
Pblp
Corn Oiljp
3 - MCIp
B(a)PIp
B(a)PIT
Dose
(Amt/kg)
-
50 mg
3 ml
20 mg
20 mg
10 mg
Time of Administration
(hr) Pre-Sacrifice
-
72,48 and
24
24
24
once/wk x
24
5, 24
IT - Intratracheal
IP - Intraperitoneal
37
-------
TABLE 5.
INFLUENCE OF ENZYME INDUCERS ON TOTAL METABOLITE APPEARANCE IN THE BLOOD
Pretreatment
Control Tp
PbIP
Corn Oi!Tp
3 - MCIp
B(a)PIp
B(a)Pn
N
9
4
5
4
5
5
Total Rate of Appearance
(ng/hr/g lung - S. E. )
256 - 38
162 ± 22
518 t 97*
836 ± 343
1718 ± 287**
1290 ± 114**
IT - Intratracheal
IP Intraperitioneal
* P = 0.05
** P = 0.01 by Student-Newman-Keuls Test
38
-------
TABLE 6.
INFLUENCE OF ENZYME INDUCERS ON THE METABOLISM OF BENZO(a)PYRENE ON THE IPL
RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animals
Control BaPIT Bapjp
BaP BaP BaP
9 5 5
Total rate of appearance
of metabolites in blood
(ng/hr/g lung - s.E.)
Metabolic pattern in
blood (% ± S.E.)
256 ± 38
1290 * 114a 1718 ± 287'
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
Monohydroxylated
Diones
Nonextractables
6.
14.
3.
9.
10.
54.
5 ±
5 ±
4 ±
7 ±
7 t
5 ±
0.
3.
0.
1.
1.
5.
9
4
6
1
8
4
5.
32.
1.
5.
2.
51.
4 ±
8 ±
8 ±
9 ±
8 ±
8 ±
2.6
8.6b
1.1
2.3b
2.1b
6.3
8.
27.
3.
13.
10.
36.
4 ±
9 ±
8 *
0 ±
6 *
3 ±
0.
4.
0.
3.
1.
4.
7
5
9
5
8
7
a. P - 0.01
b. P = 0.05 by Student-Newman-Keuls Test
All three columns compared to each other. All metabolites separated
by tic.
39
-------
TABLE 7.
COMPARISON HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
BaP
4
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S.E.)
Metabolic pattern in
blood (% ± S.E.)a
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
1 ,6-6,12-3,6-quinone
4,5 epoxide^
9,6-OH
7-OH
3-OH
S Metabolite
Nonextractable
HPLC
334^40
20.1-6.4
2.8-0.4
2.7^0.6
1.4-0.6
2.3-0.6
2.3-1.0
2.1-0.9
9.4^3.8
54.3-7.7
tic
364^45
18.3-7.5
2.2-1.0
10.3^2.7
3.6^0.8
6.3-2.7
59.2-9.9
Metabolite pattern values expressed as percent of total rate of appearance
of metabolites in blood - S.E.
14C counts appear under peak.
C7-OH and 3-OH collected together.
40
-------
TABLE 8.
COMPARISON OF HPLC AND tic DATA
Pretreatment
BaP
IT
Appearance of
all metabolites
in blood at
60 minutes +
(ng/g lung - S.E.)
HPLC
1505 - 157
tic
1431 - 153
Metabolite Pattern
(% t S.E.)W
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-quinone
4,5-epoxide-y
9 & 6-OH
3 & 7-OH
S metabol ite
nonextractable
30.1 - 5.1 37.7 - 5.9
3.2 - 0.2 1.4 - 0.4
3.6 - 0.3 7.6 - 1.5
1.5 - 0.1
3.2 - 0.9
5.8 - 0.8
2.5 - 0.4
3.7 - 0.2
3.3 - 1.0
6.7 - 1.3
5.1 - 0.8
41.1 - 4.4 43.3 - 4.5
wMetabolite pattern values expressed as % of appearance of all
metabolites in blood at 60 minutes - standard error.
Y14c counts appear under this peak.
n - 4
41
-------
TABLE 9.
COMPARISON OF HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
HPLC
Appearance of
all metabolites
in blood at 1624 - 227
60 minutes +
(ng/g lung - S.E. )
BaPIp
BaP
5
tic
1741 - 378
Metabolite Pattern
(% ± S.E.)W
9,10-dihydrodiol 30.4 - 6.0
4,5-dihydrodiol 3.8 - 0.5
7,8-dihydrodiol 3.1 - 0.5
4,5-quinone 1 .4 - 0.3 ~
3,6-,1,6-,6,12-quinone 2.8-0.5
4,5-epoxidey 2.0 - 0.5
9 & 6-OH 3.9 +- 1.5 ~
3 & 7-OH 4.2 - 1.0
nonextractable 37.0 - 4.4
S metabolite 11.7 - 3.2
27.5 - 4.2
4.1 - 0.7
8.7 - 1.2
10.6 - 1.9
- 13.0 - 3.5
34.0 - 4.1
wMetabolite pattern value expressed as % of appearance of all metabolites
in blood at 60 minutes ± standard error.
y
14C counts appear under this peak.
42
-------
b. Influence of Various PT450 Enzyme Inducers
BaP is a better enzyme inducer than 3MC, as indicated by the total
rate of metabolism (Table 10). The metabolic pattern indicates that
both 3MC and BaP have similar profiles which are different from the
corn oil control. There appears to be more 9,10-dihydrodiol, diones,
and phenols and less nonextractables than the control corn oil. HPLC
data for corn oil (Table 11) at 60 minutes suggests that the 7,8-di-
hydrodiol may be mostly the unknown S metabolite (see methods).
These data suggest that most of metabolites formed by P^SO enzyme
inducers have similar metabolic pathways and that more polar material
is excreted into the blood stream. The metabolic turnover rate is
faster and, therefore, the increased amount of epoxides that are
formed as intermediates rearrange or isomerize to phenols and oxidize
to quinones or hydrate to the 9,10-dihydrodiols by epoxide hydrase
action. This shunting to other pathways produces a smaller percentage
of bound material (nonextractables) (64).
c. Influence of Pj450 Enzyme Inducer on BaP Metabolism
Phenobarbital does not induce the aryl hydrocarbon hydroxylase
enzyme system (P^BO) as seen in Table 12 (64). Corn oil, however, is
significantly different from the control which indicates that corn
oil does induce the enzyme system. The metabolic profiles for corn oil
and the control are similar, showing only slightly fewer phenols and
quinones for corn oil. Phenobarbital, on the other hand, produces an
increased quinone formation which is consistent with lack of P^BO
induction.
d. Distribution of BaP and Metabolites in Tissues at 180 Minutes
in the IPL Following BaP Pretreatment
there is less unmetabolized BaP remaining in the IPL with either
BaPj-r or BaPjp pretreatment than their appropriate controls. This is
consistent with an increase in enzyme activity in each case due to
the BaP (Table 13 and 14).
There is a relative increase in the metabolic material and a
decrease in BaP in the blood at 180 minutes after BaPjp or BaPjy
pretreatment compared to their appropriate controls. This is reflected
in a large increase in the 9,10-dihydrodiol and the nonextractables.
Smaller increases are observed for the phenols and quinones after BaPjp
pretreatment.
Similar results are obtained for the distribution in the lung.
There is a relative increase in the metabolite material and a decrease
in BaP in the lung at 180 minutes after BaP pretreatment compared to
controls. This is reflected in a large increase in the nonextractables
43
-------
after IT pretreatment and an increase in nonextractables and phenols
after IP pretreatment. There is very little metabolite in the macro-
phage under either pretreatment. Also, there is a decrease in the
relative amount of BaP in BaP pretreatments compared to controls.
This is reflected in a slight increase in phenols, quinones, and non-
extractables in both cases.
The relative amounts of metabolite in trachea bronchi decrease
slightly after BaPIT pretreatment and increase slightly after BaPjp
pretreatment as compared to their controls. There is a large decrease
in the relative amounts of BaP given in the trachea bronchi. These
data are reflected in an increase in nonextractables for IT pretreat-
ment, and an increase in the dione fraction and decrease in nonextract-
ables for IP pretreatment. Lastly, the increase in the relative amounts
of metabolite in the washout are indicated by large increases in the
nonextractable material.
Summary
With the addition of BaPjp or BaPjj as a pretreatment, there are
large increases in rate of appearance of metabolites in the blood and
large changes in the distribution of BaP and its metabolites in the
blood and lung. With concomitant increases in total metabolite in
blood and lung, there are corresponding decreases of BaP in these
tissues. These major changes are consistent with increases in non-
extractables in blood and lung and with increases in 9,10-dihydrodiol
in the blood. Small increases and decreases of BaP and metabolite in
the macrophage, washout, and trachea bronchi are also observed in
relative changes in nonextractables and quinones or phenols. This
suggests that as more intermediate epoxides are formed at a faster
rate, they are converted into conjugates of BaP, or bound to macro-
molecules. Under both pretreatments, as the pathway becomes saturated,
the epoxide hydrase converts the 9,10-epoxide to the 9,10-dihydrodiol
and excretes it into the blood. Rearrangements and/or isomerization
converts some intermediates to phenols and quinones after BaPjp pre-
treatment. This last statement also suggests differences due to
route of administration which in turn reflects differences in enzyme
activities.
44
-------
TABLE 10.
INFLUENCE OF ENZYME INDUCERS ON THE METABOLISM OF BENZO(a)PYRENE ON THE IPL
RATE AND PATTERN OF METABOLISM IN THE BLOOD
P re treatment
No.
IPL
of Animals
Corn Oiljp 3-MCIp
BaP BaP
5 4
BaPIp
BaP
4
Total rate of appearance
of metabolites in blood 518 ± 97 836 ± 343 1718 * 287
(ng/hr/g lung - S.E.
Metabolic pattern in
blood (% ± S.E.)
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
Monohydroxylated
Diones
Nonextractables
17
15
1
2
6
56
.9
.4
.2
.7
.5
.3
t 4.
- 4.
±0.
- 1.
+ 2.
t 7.
9
9
5
1
9
8
5.
33.
4.
14.
11.
32.
4 +
3 +
3+-
0 -
7±
6 +
1.
2.
0.
2.
3.
2.
oa
ob
8
4a
8
7
8.
27.
3.
13.
10.
36.
4± 0.
9-4.
8 + 0.
0±3.
6 + L
3^4.
7a
5
9
5b
8
7
a. P - 0.01
b. P = 0.05 by Student-Newman-Keuls Test
All three columns compared to each other. All metabolites separated
by tic.
45
-------
TABLE 11.
COMPARISON OF HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
Appearance of
all metabolites
in blood at
60 minutes +
(ng/g lung - S.E. )
Metabolite Pattern
(7 ± S E )w
\ '" o • c. . ;
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-,l ,6-,6,12-quinone
4,5-epoxidey
9 & 6-OH
3 & 7-OH
nonextractable
S metabolite
Corn Oil *„
BaP
5
HPLC tic
581 - 63 647 - 90
16.5 - 5.5 10.7 - 2.5
2.8 - 1.1 1.3 - 0.5
2.1 - 0.6 25.4 - 9.7
1.8-0.4
5.9 - 2.0 - 8.9 - 4.0
1.9 - 0.5
+
2.8 - 0.8 .
, _ 2.7 - 1.0
2.4 - 0.6
52.3 - 9.4 51.0 - 11.7
10.9 - 2.0
'Metabolite pattern value expressed as % of appearance of all
metabolites in blood at 60 minutes - standard error.
counts appear under this peak.
46
-------
TABLE 12.
INFLUENCE OF ENZYME INDUCERS ON THE METABOLISM OF BENZO(a)PYRENE ON THE IPL
RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animals
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S.E. )
Metabolic pattern in
blood (% ± S.E.)
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
Monohydroxylated
Diones
Nonextractables
Control Pheno Corn Oil jp
BaP BaP BaP
9 4 5
256 - 38 162 - 22 518 - 97b
6.5 - 0.9 6.9 - 2.0 17.9 - 4.9a
14.5 - 3.4 4.8 - 1.0C 15.4 - 4.9
3.4 - 0.6 3.1 - 1.0 1.2 - 0.5b
9.7 - 1.1 6.3 - 1.1C 2.7 - l.la
10.7 - 1.8 21.9 - 4.4b 6.5 - 2.9
54.5 - 5.4 59.1 - 6.5 56.3 - 7.8
a. P - 0.01
b. P = 0.05
c. P = 0.10 (by Student-Newman-Keuls Test)
All three columns compared to each other. All metabolites separated
by tic.
47
-------
TABLE 13.
ENZYME INDUCERS
% OF TOTAL BaP AND TOTAL METABOLITE REMAINING IN EACH TISSUE
AT 180 MINUTES - S.E.
Pretreatment
IPL
No. of Animals
% of Unmetabolized
BaP
% of Total Compound
as Metabolite in
Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound
as BaP in Tissue
Blood
TB
MAC
WO
Lung
None
BaP
3
65.
15.
3.
0.
3.
12.
12.
10.
8.
2.
30.
3
5
7
3
3
0
7
4
7
5
9
± 4.
,
- 5.
,
- 1.
,
- 0.
,
- 2.
,
- 1.
,
- 1.
,
- 2.
,
- 7.
± 1.
i
- 3.
3
2
2
1
3
9
4
7
4
1
6
BaPIT
BaP
3
21.9 - 3.1
+
41.5 - 7.5
4-
1.3 - 0.5
4.
0.8 - 0.2
4-
6.3 - 0.5
4-
28.0 - 1.4
-i-
1.9 - 0.2
-t-
1.5 - 0.5
_i_
4.7 - 0.6
2.4 ± 0.6
4.
11.5 - 3.0
Corn Oil
BaP
2
60.9 ±
+
14.5 -
4-
3.4 ±
4-
0.9 ±
+
2.6 ±
4.
17.6 ±
4.
15.8 -
4.
5.4 ±
4-
3.8 ±
5.2 ±
_l_
30.6 -
3.
2.
0.
0.
0.
0.
5.
1.
0.
3.
4.
4
5
1
3
4
8
6
8
4
9
7
23.
39.
5.
0.
8.
21.
1.
6.
1.
2.
12.
BaPIp
BaP
2
5±
-i-
7 -
4-
9 ±
4.
6 ±
4-
9 -
4.
4 ±
4-
5 ±
4.
2 ±
_i_
0 ±
1 ±
_i_
6 ±
2.5
17.9
4.6
0.5
7.2
8.0
1.0
3.0
0.7
0.4
0.7
48
-------
TABLE 14.
DISTRIBUTION PATTERN OF BaP + METABOLITES IN EACH TISSUE
Pretreatment
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
Control
1.2-0.1
2.9-1.5
0.9-0.4
10.1-0.6
1.1-0.3
5.3-0.9
78.5-2.7
LUNGX
IT
5.4-0.7
2.7-0.6
0.9-0.3
3.5-1.4
3.7-0.7
43.9-4.6
39.8-7.4
Corn Oil
3.5-0.1
1.0-0.3
0.3-0.0
2.2-1.5
1.2-0.2
13.2-1.9
78.5-3.4
BaP
bahIP
4.5-1.3
5.4^2.7
0.6-0.0
11.9-6.1
4.9-1.4
26.7-0.5
45.9-10.9
y +
Based on % total activity at 180 minutes in each tissue - S.E.
Control, BaPIT, Corn Oil and BaPjp - 3,3,2,2 animals respectively.
Control and BaP,-,- comparable.
Corn Oil and BaPjp comparable.
(continued)
49
-------
TABLE 14.
(continued)
Pretreatment
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxyl ated
diones
nonextractable
BaP
Control
2.8-0.5
6.5-3.8
1.2-0.7
2.5-0.9
1.3-0.1
18.6^2.5
4-
67.0-6.5
180 BLOODX
BaPIT
6.4-1.4
17.4-2.5
1.9-1.2
3.3-0.6
1.9-0.4
61.5-2.5
4-
7.6-1.4
Corn Oil
2.3-0.1
2.3-1.1
0.8-0.5
2.8-0.7
1.1-0.3
21.9-4.2
4-
68.9-4.1
BaPIp
6.5-0.6
14.8-5.9
1.0-0.3
9.1-5.4
8.9-3.4
55.4-16.5
4.
4.2-1.4
X +
Based on % total activity at 180 minutes in each tissue - S.E.
Control, BaPIT, Corn Oil and BaPIp - 3,3,2,2 animals respectively.
Control and BaPyT comparable.
Corn Oil and BaPjp comparable.
(continued)
50
-------
TABLE 14.
(continued)
Pretreatment
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
Control
1.4-0.4
4.
0.9-0.2
0.2-0.2
0.9-0.3
0.5-0.2
,
1.9-1.0
4.
9.42-2.2
MACROPHAGEX
BaPIT
1.4-0.3
4.
0.9-0.2
0.1-0.0
1.3-0.6
2.4-1.0
4-
3.5-0.9
4.
90.3-2.5
Corn Oil
2.6-0.9
4-
4.2-3.0
0.0-0.0
1.0-0.3
0.4-0.1
4-
2.2-0.7
4-
89.6-4.1
BaPIp
2.6-0.6
4-
2.3-0.6
0.7-0.1
5.6-2.9
8.0-7.6
4-
8.4-4.5
4-
72.5-5.9
X +
Based on % total activity at 180 minutes in each tissue - S.E.
Control, BaPIT, Corn Oil and BaPjp - 3,3,2,2 animals respectively
Control and BaPyT comparable.
Corn Oil and BaPp comparable.
(continued)
51
-------
TABLE 14.
(continued)
Pretreatment
7,8-dyhydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
Control
5.0-1.8
10.8-8.3
0.8-0.6
2.7-2.0
2.1-1.3
17.5-6.6
61.0-7.7
WASHOUTX
BaPIT
5.5^1.8
7.1-1.2
0.6-0.4
3.8-1.1
3.8-1.3
39.6-4.6
39.6^2.2
Corn Oil
4.5-1.5
2.5-1.1
0.4^0.1
1.6-1.1
0.7-0.1
18.9-16.3
71.5-15.5
BaPIp
3.5-0.8
7.3-4.8
0.7-0.1
7.4-1.4
2.1-0.0
39.8-19.7
39.2-26.6
xBased on % total activity at 180 minutes in each tissue - S.E.
Control, BaPIT, Corn Oil and BaPIp - 3,3,2,2 animals respectively.
Control and BaP.-,- comparable.
Corn Oil and BaPTp comparable.
(continued)
52
-------
TABLE 14.
(continued)
TRACHEA BRONCHI'
Pretreatment
Control
Corn Oil
7,8-dyhydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
0.8-0.2
2.2-1.2
0.9-0.5
3.4-1.8
2.1-1.4
12.5-4.1
79.4-7.4
4.4-0.7
3.4-0.8
0.5-0.2
1.4-0.7
3.2-2.6
24.8-11.5
62.2-10.1
1.5-0.9
0.7-0.7
0.5-0.2
1.1-0.0
0.7-0.7
17.4-10.7
78.1-8.2
2.3-0.3
3.1-0.8
0.7^0.6
4.0-2.2
11.9-6.2
62.4-2.6
65.5-11.4
X +
Based on % total activity at 180 minutes in each tissue - S.E.
Control, BaPTJ, Corn Oil and BaPIp - 3,3,2,2 animals respectively.
Control and BaPIT comparable.
Corn Oil and BaPIp comparable.
53
-------
2. Effects of Particulate
a. Influence of Particulate Administered to IPL on BaP
Metabolism
1) Rate of Metabolism
The crude air particulate (or ferric oxide) acts to inhibit the
rate of metabolism (Table 15) of BaP slightly. This can be due to
either a biochemical effect or a physical effect, i.e. particulate
engulfed more readily by macrophages, and therefore, rendering the BaP
less biologically available for metabolism over the time of study.
However, there is not a significant differency between CAP (or ferric
oxide) and BaP and BaP alone on the rate of metabolism of BaP on the
IPL. The metabolic pattern shows a marked increase in 7,8- and 9,10-
diols and a decrease in the nonextractable or polar and monohydroxy
and quinone metabolites. This indicates an inhibition of the particu-
late on the multiple enzymes (17,28,37,48,71) which is consistent with
diol epoxide formation and a decrease in hydroxylation and/or isomeri-
zation and polar (nonextractable) conjugation pathways. The mechanism
for the decrease in the nonextractable or polar metabolite is not
understood at present.
The HPLC data generally supports the TLC data (Table 16). There
are a few differences that may be due to 1) the techniques involved
and 2) the values at 60 minutes on HPLC versus the values obtained
from the slope of the curve for TLC (25,55).
2) Distribution of BaP and Its Metabolites in Tissue at
180 Minutes
There is a relative decrease of BaP in the blood at 180 minutes
compared to control (Table 17 & 18), while the lung and trachea bronchi
show a relative increase in metabolite and a decrease in BaP. These
results are consistent with an increase in the nonextractables. The
macrophage and washout both show increases in the relative amounts of
the BaP metabolite content. The only changes that are observed in
their metabolite profiles are the 7,8 and 9,10-diols and the nonextract-
ables for the washout fraction.
Summary
The particulate inhibits the rate of appearance of metabolites of
BaP observed in the blood. This can be explained by the relative in-
crease of BaP and metabolites in the macrophage and washout with
corresponding decrease of BaP in the lung and trachea bronchi. The BaP
adsorbed particulate can be engulfed more readily by macrophages and,
therefore, BaP is less biologically available for metabolism or is
leached more slowly from the lung. However, at the end of 180 minutes
54
-------
the relative amount of BaP metabolized by CAP plus BaP is greater than
the control. This is consistent with an increase of metabolite in
lung and trachea bronchi, as well as washout and macrophage. This
then might suggest that the CAP, which contains a variety of PAHs and
metals, might induce the enzyme system slightly after a period of time
for equilibration or that the macrophage metabolize BaP but dealy the
excretion of these metabolites into the blood.
55
-------
TABLE 15.
INFLUENCE OF PARTICULATES ADMINISTERED TO IPL OR BaP METABOLISM
RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL BaP BaP + CAP3
No. of Animals 9 5
Total rate of appearance , +
of metabolites+in blood 256 - 38 156 - 42
(ng/hr/g lung - S.E.)
Metabolic pattern in
blood (% ± S.E.) b
7,8-dihydrodiol 6.6 - 0.9 19.1 - 4.4C
9,10-dihydrodiol 15.4 - 4.0 28.3 - 7.9
4,5-dihydrodiol 3.3-0.6 3.0-1.3
Monohydroxylated 9.7-1.1 5.1 - 1.4e
Diones 10.6 - 1.8 5.2 - 2.6
Nonextractable 54.4 - 5.4 39.3 - 13.8
All metabolites separated by tic.
al mg/kg
Metabolite pattern expressed as percent of total rate of appearance
of metabolites in blood - S.E.
CP - 0.01
dP = 0.05
eP = 0.1 (Student-Newman-Keuls Test)
56
-------
TABLE 16.
COMPARISON OF HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
None
BaP + CAP
5
Appearance of
all metabolites
in blood at
60 minutes
(ng/g lung - S.E.)
HPLC
208 - 18
tic
203 - 34
Metabolite Pattern
("/ S F ^
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-,l ,6-,6,12-quinone
4,5-epoxide-y
9 & 6-OH
3 & 7-OH
nonextractable
S metabolite
14.9
2.3
2.8
3.0
3.4
1.4
1.9
2.8
50.6
16.8
- 6.8 25.0 - 7.5
- 0.9 1.8 - 1.6
- 0.6 11.2 - 2.8
- 0.7
- 1.0 2.8 - 1.1
±0.5
- 0.7 4.2 - 1.4
±0.9
- 8.2 54.9 - 11.7
± 3.0
"Vletabolite pattern value expressed as % of appearance of all
metabolites in blood at 60 minutes ± standard error.
^14 counts appear under this peak.
57
-------
TABLE 17.
INFLUENCE OF PARTICULATE ON THE IPL
OF TOTAL BaP AND TOTAL METABOLITE REMAINING IN EACH TISSUE
AT 180 MINUTES * S.E.
Pretreatment
IPL
No. of Animals
% Unmetabolized BaP
7o of Total Compound as
Metabol ite in Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound as
BaP in Tissue
Blood
TB
MAC
WO
Lung
BaP
3
65.3 - 4.3
15.5 - 5.3
3.7 - 1.2
0.3 - 0.1
3.3 - 2.3
12.0 - 1.9
12.7 - 1.4
10.4 - 2.7
8.7 - 7.4
7.5 - 1.1
30.9 - 3.6
BaP + CAP
2
40.0 - 26.3
16.6 - 6.7
6.2 - 2.8
1.9-1.3
13.2 - 3.3
22.1 - 9.1
1.0 - 0.8
2.3 - 1.0
17.4 - 12.2
10.9 - 9.2
8.5 - 2.5
58
-------
TABLE 18.
DISTRIBUTION PATTERN OF BaP + METABOLITES IN EACH TISSUE
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
LUNGX
None
BaP
1.2-0.1
2.9-1.5
0.9-0.4
10.1-0.6
1.1-0.3
5.3-0.9
78.5-2.7
None
BaP+CAP
6.1-0.5
3.2^0.2
1.3-0.2
3.4-0.7
1.3-1.0
24.7-2.1
60.0-3.2
180
None
BaP
2.8-0.5
6.5-3.8
1.2-0.7
2.5-0.9
1.3-0.1
18.6-2.5
67.0-6.5
BLOODX
None
BaP+CAP
8.0-5.8
5.6-3.4
0.9-0.6
2.2-1.8
1.5-1.3
X59.7-33.7
X22.1-20.8
X +
Based on % of total activity at 180 minutes in each tissue - S.E,
Number of animals 3 & 2 respectively.
(continued)
59
-------
TABLE 18.
(continued)
MACROPHAGEX
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
None
BaP
1.4-0.4
+
0.9-0.2
0.2-0.2
4-
0.9-0.3
4.
0.5-0.2
1.9-1.0
4-
94.2-2.2
None
BaP+CAP
1.5-0.5
•
0.3-0.2
0.1-0.0
4.
0.2-0.1
4.
0.2-0.1
1.4-0.3
4-
96.3-1.3
WASHOUTX
None
BaP
5.0-1.8
•
10.8-8.3
0.8-0.6
,
2.7-2.0
4.
2.1-1.3
17.5-6.6
4.
61.0-7.7
None
BaP+CAP
X28.4-19.6
i
5.3-0.5
0.9-0.3
,
3.2-2.1
4.
2.4-0.6
0.00
Y 4-
X59. 9-22.1
xBased on % of total activity at 180 minutes in each tissue - S.E.
Number of animals 3 & 2 respectively.
(continued)
60
-------
TABLE 18.
(continued)
TRACHEA BRONCHIX
Pretreatment None None
IpL BaP BaP+CAP
7,8-dihydrodiol 0.8-0.2 3.5-1.2
9,10-dihydrodiol 2.2-1.2 2.6-1.7
4.5-dihydrodiol 0.9^0.5 0.4-0.3
monohydroxylated 3.4-1.8 0.3*0.3
diones 2.1*1.4 3.5*1.5
nonextractable 12.5*4.1 X26.7*16.1
BaP
79.4*7.4 63.1*21.0
x +
Based on % of total activity at 180 minutes in each tissue S.E.
Number of animals 3 & 2 respectively.
61
-------
b. Influence of BaP Pretreatment and Particulate Administered
on IPL on BaP Metabolism
1 ) Rate of Metabolism
BaP pretreatment increases significantly the rate of metabolism
as compared to the corn oil control. BaP pretreatment and CAP or
ferric oxide in perfusion, however, decrease the rate of metabolism
significantly as compared to BaPIP control (Table 19) and is comparable
to the corn oil control. The metabolic effect of the BAP may be due to
an increase in the rate of action of the macrophage or the slow physical
release of BaP. The rate of metabolism due to the particulate is not
influenced by pretreatment with enzyme inducers. The pattern due to
the particulate indicates a trend toward glutathione transferase and/or
epoxide hydrase and away from isomerization and/or polyhydroxylation.
This is reflected in an increase in the polar or nonextractable and 7,8-
and 9,10-diol metabolites versus diones and monohydroxylated metabolites.
Secondly, the particulate increases the nonextractable or polar material
versus the diols, i.e. possible BaP conjugation versus epoxide hydrase
and hydration. Lastly, the metabolite profile for BaPjp followed by
BaP plus CAP on the IPL is very similar to the corn oil pretreatment
alone, which further indicates the effect of CAP on this type of pre-
treatment. As indicated, HPLC data supports tic data (Table 20) rather
well. The 7,8-dihydrodiol and the S metabolite on HPLC chromatograph
together on tic (25,55).
)'(•-",
2) Distribution of BaP and Its Metabolites in Tissue at
180 Minutes
There is a large increase in the relative amount of metabolites
and a corresponding relative decrease of BaP in both the blood and
lung for the BaPjp pretreatment compared to its control (Fig. 21 & 22).
This also is consistent with a decrease in the relative amount of total
BaP left in perfusion at 180 minutes. The results for the BaPjp pre-
treatment followed by BaP plus CAP experiments are similar to BaPjp
pretreatment experiments. The results for the blood are reflected in
an increase in the excretion of the nonextractables, as well as the
9,10-dihydrodiol and phenols. There are some differences, however,
between the two experimental groups in the lung; there is a larger
amount of phenol and nonextractable in BaP pretreatment experiments
than in the CAP experimental group. This can be attributed to the
fact that the BaP may be more readily available for metabolism than
in the case involving CAP plus BaP.
This last fact is consistent with the relative increase in qui-
nones, phenols and nonextractables and the decrease in BaP in the
macrophage for BaPjp pretreatment. These results are not seen when CAP
is introduced in the system. CAP would appear to enhance the action of
the macrophage.
62
-------
There appears to be minor changes in the relative amounts of BaP
and metabolite in washout and trachea bronchi for both experimental
groups. In both cases the relative amounts of nonextractables are
increased in the washout with a larger increase observed when CAP is
introduced. In the trachea bronchi there is a decrease in the non-
extractables and a relative increase in phenols for BaPjp pretreatment
compared to its control. When CAP is introduced a higher percentage of
BaP is found which is consistent with the fact that BaP is not available
for metabolism or that clearance of BaP is slowed by CAP.
63
-------
TABLE 19.
INFLUENCE OF BaP PRETREATMENT AND PARTICULATE ADMINISTERED ON IPL
ON BaP METABOLISM RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment Corn Oil^a
M IP,L. . . BaP BaP BaP + CAPC
No. of Animal s 555
Total rate of appearance , , ,
of metabolites in blood 466 - 94 1718 - 287 414 - 95e
(ng/hr/g lung - S.E.)
Metabolic pattern in
blood (% - S.E.)d
7,8-dihydrodiol 17.9 - 0.9f 8.4-0.7 11.8-0.6a
9,10-dihydrodiol 15.3 - 4.9 27.9 - 4.5 18.6 + 2.7
4,5-dihydrodiol 1.2-0.59 3.8-0.9 2.6-1.6
Monohydroxylated 2.7 - l.l9 13.0 - 3.5 3.5 - l.O9
Diones 6.5 - 2.9 10.6 - 1.8 2.3 - 0.6e
Nonextractable 56.3 ^ 7.8 36.3 - 4.7 61.1 - 3.6g
Columns 1 & 3 compared with column 2. All metabolites separated by tic.
a3 ml/kg.
20 mg/kg (24 hrs. before sacrifice).
1 mg/kg.
Metabolite pattern values expressed+as percent of total rate of
appearance of metabolites in blood - S.E.
eP = 0.01
fP = 0.1
9P = 0.05 (Student-Newman-Keuls Test)
64
-------
TABLE 20.
COMPARISON OF HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
BaPIp
BaP + CAP
4
Appearance of
all metabolites
in blood at
60 minutes +
(ng/g lung - S.E.)
Metabolite Pattern
(°l S F 1W
\ h - 3 . L. )
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-,l,6-,6,12-quinone
4,5-epoxide^
9 & 6-OH
3 & 7-OH
nonextractable
S metabolite
HPLC
424 - 88
16.6 - 3.2
3.1 - 0.4
2.2 - 0.5
2.3 - 0.5
2.0 - 0.6
0.8 - 0.3
1.1 - 0.5
2.2 - 0.5
60.6 - 4.5
9.0 - 1.4
tic
453 - 99
20.1 - 2.5
2.9 - 1.7
13.5 - 2.9
3.4 - 1.7
3.4 - 1.1
56.5 - 5.1
wMetabolite pattern value expressed as % of appearance of all
metabolites in blood at 60 minutes - standard error.
^14r counts appear under this peak.
65
-------
TABLE 21.
INFLUENCE OF BaP PRETREATMENT
% OF TOTAL BaP AND TOTAL METABOLITE REMAINING IN EACH TISSUE
AT 180 MINUTES - S.E.
Pretreatment
IPL
No. of Animals
% Unmetabolized BaP
% of Total Compound
as BaP in Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound
as BaP in Tissue
Blood
TB
MAC
WO
Lung
Corn Oil
BaP
2
60.9 - 3.4
14.5 - 2.5
3.4 - 0.1
0.9 - 0.3
2.6 - 0.4
17.6 - 0.8
15.8 - 5.6
5.4 - 1.8
3.8 - 0.4
5.2 - 3.9
30.6 - 4.7
BaPIp
BaP
2
23.5 - 2.5
39.7 - 17.9
5.9 - 4.6
0.6 - 0.5
8.9 - 7.2
21.4 - 8.0
'V'
1.5-1.0
r6.3 - 3.0
1.0 - 0.7
2.1 - 0.4
12.6 - 0.7
BaPIp
BaP + CAP
2
25.2 - 5.9
41.2 - 14.7
3.8 - 1.4
0.7 - 0.1
4.4 - 1.9
24.5 - 14.2
1.4 i 0.7
5.3 - 1.5
3.4 - 0.5
0.2 - 0.0
14.8 - 6.8
Column 1 compared to Column 2.
Column 3 compared to Column 2.
-------
TABLE 22.
% DISTRIBUTION PATTERN OF BaP + METABOLITES IN EACH TISSUE
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
LUNGX
Corn Oil ,p
BaP
3.5-0.1
1.0-0.3
0.3-0.0
BaPIp
BaP
4.5-1.3
5.4-2.7
0.6-0.0
BaPIp
BaP+CAP
2.6-0.0
4.5-3.8
0.7-0.7
monohydroxylated 2.2-1.5 11.9-6.1 0.9-0.6
diones 1.2-0.2 4.9-1.4 0.2-0.2
nonextractable 13.2-1.9 26.7-0.5 18.9-0.4
BaP 78.5-3.4 45.9-10.9 72.1-3.1
x +
Based on % total activity at 180 minutes in each tissue - S.E.
Two (2) animals per experiment respectively.
Corn Oiljp compared to BaPIp.
& BaP + CAP on IPL compared to BaPjp.
(continued)
67
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxyl ated
diones
nonextractable
BaP
TABLE 22.
(continued)
180 BLOODX
Corn Oil Ip
BaP
2.3-0.1
2.3-1.1
4-
0.8-0.5
2.8-0.7
1.1-0.3
4.
21.9-4.2
68.9-4.1
BaPIp
BaP
6.5-0.6
14.8-5.9
4.
1.0-0.3
,9.1-5.4
8.9-3.4
4.
55.4-16.5
4.2-1.4
BaPIp
BaP+CAP
7.8-2.0
11.7-2.7
4.
oToo
11.6-1.1
1.2-0.2
_L
56.2-1.6
11.5-1.7
xBased on % total activity at 180 minutes in each tissue - S.E.
Two (2) animals per experiment respectively.
Corn Oiljp compared to BaPTp.
BaPIp & BaP + CAP on IPL compared to BaPIp.
(continued)
68
-------
TABLE 22.
(continued)
MACROPHAGE*
Pretreatment Corn Oiljp BaPIp BaPIp
IPL BaP BaP BaP+CAP
7,8-dihydrodiol 2.6-0.9 2.6-0.6 1.5-0.4
9,10-dihydrodiol 4.2-3.0 2.3-0.6 1.3-1.0
4,5-dihydrodiol 0.0-0.0 0.7-0.1 0.1-0.1
monohydroxylated 1.0-0.3 5.6-2.9 0.1-0.0
diones 0.4-0.1 8.0-7.6 0.3-0.2
nonextractable 2.2-0.7 8.4-4.5 2.3-0.7
BaP 89.6-4.1 72.5-5.9 94.4-1.9
y +
Based on % total activity at 180 minutes in each tissue - S.E.
Two (2) animals per experiment respectively.
Corn Oiljp compared to BaPjp.
p & BaP + CAP on IPL compared to BaPIp.
(continued)
69
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxlated
diones
nonextractable
BaP
TABLE 22.
(continued)
WASHOUTX
Corn Oil ,p
BaP
4.5-1.5
2.5-1.1
0.4-0.1
1.6-1.1
0.7^0.1
18.9-16.3
71.5-15.5
BaPIp
BaP
3.5-0.8
! 7.3-4.8
', 0.7-0.1
1
; 7.4-1.4
2.1-0.0
39.8-19.7
39.2-26.6
BaPIp
BaP+CAP
4.2-1.9
3.8-2.3
0.4-0.1
0.4-0.0
1.5-0.8
68.8-7.0
20.8-6.8
xBased on % total activity at 180 minutes in each tissue - S.E.
Two (2) animals per experiment respectively.
Corn Oiljp compared to BaPTp.
& BaP + CAP on IPL compared to BaPIp.
(continued)
70
-------
TABLE 22.
(continued)
TRACHEA BRONCHIX
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
Corn Oil Ip
BaP
1.5-0.9
0.7-0.7
0.5-0.2
1.1-0.0
0.7-0.7
17.4-10.7
78.1-8.2
BaPIp
BaP
2.3-0.3
3.1-0.8
0.7-0.6
4.0-2.2
11.9-6.2
12.4-2.6
65.5-11.4
BaPIp
BaP+CAP
1.9-1.1
1.3^0.9
0.6-0.3
0.5-0.3
0.8-0.7
9.7-2.0
85.1-1.4
Based on % total activity at 180 minutes in each tissue - S.E.
Two (2) animals per experiment respectively.
Corn Oiljp compared to BaPjp.
& BaP + CAP on IPL compared to BaPIp.
71
-------
Summary
When BaP Is given IP to the whole animal, the rate of appearance
of metabolites is increased significantly,, compared to its control.
However, when CAP is introduced with BaP on the perfusion the rate of
appearance of metabolites is decreased. The rate of appearance and
the metabolite profile are similar to the corn oil pretreatment, which
indicates that CAP negates the effect of pretreatment. However, as the
perfusion proceeds through to 180 minutes, more BaP is metabolized,
i.e. more BaP is available by leaching from CAP. In fact, the profiles
are similar for the two experimental groups which suggests that after
equilibration for a period of time, more of the BaP adsorbed on CAP is
available for metabolism. This is reflected in larger amounts of non-
extractables and 9,10-diol excreted into the blood with corresponding
decreases of BaP. However, there are still some minor differences at
180 minutes in that slightly more BaP and less metabolites are found in
the macrophage, lung, and trachea bronchi after introduction of CAP.
With additional time, more BaP may become .available for metabolism, and
therefore, these differences could disappear. Lastly, it should be
mentioned that the CAP inhibits induced enzymes but not the basal
enzymes.
c. Influence of Parti cul ate Pretreatment on BaP Metabolism
1 ) Rate of Metabolism
Pretreatment with particulate causes a significant increase in
the rate of metabolism (Table 23) versus i^ts control. This suggests
that the particulate may influence enzyme activity. This, of course,
is especially reflected in CAP pretreatment due to the presence of other
PAHs in the mixture. The metabolite profile shows a decrease in the
nonextractable or polar material and monohydroxy compounds and an
increase in the 7,8- and 9,10-diol formation. This reflects an
increase in epoxide hydrase activity and a decrease in polar (non-
extractable) conjugation.
When BaP adsorbed on CAP is added as pretreatment, the CAP acts
to greatly inhibit the enzyme inducing ability of the BaP (results,
Section la). The rate of appearance of metabolites is increased, how-
ever, for this set of experiments compared to its control which indi-
cates a combined effect of BaP and CAP- The metabolite profile shows a
decrease in the 7,8- and 9,10-diol formation and a small increase in
the nonextractable material. This reflects a slight decrease in
epoxide hydrase activity and an increase in the nonextractable con-
jugation.
The HPLC supports the tic data (Table* 24 & 25) for CAP alone and
BaP adsorbed on CAP as pretreatments (25,55).
72
-------
2) Distribution of BaP and Its Metabolites in Tissues at
180 Minutes
CAP given as a pretreatment (based on one experiment at 180
minutes) to the whole animal increases the amount of metabolite found
in tissues at 180 minutes (Table 26 & 27). There is a corresponding
relative increase of metabolite found in the blood and lung and a
relative decrease in BaP. This is reflected in an increase in the
9,10- and 7,8-diols and a decrease in the BaP content in both tissues.
The nonextractables increase in the lung and decrease in the blood
compared to the control. There are some small relative increases of
metabolite in the trachea bronchi and macrophage and corresponding
decreases of BaP in these tissues while there are slight relative
decreases in metabolite and BaP in washout compared to the control.
These data are consistent with significant increases of 7,8- and
9,10-diols and quinones in macrophage and washout, increases of the
diones in the trachea bronchi, and a significant increase of the
nonextractables in the washout. These data suggest that CAP given as
a pretreatment acts as an enzyme inducer; an increase in the rate of
metabolism of BaP is reflected in an increase of diol formation in
the tissues of the lung and smaller increases in the nonextractables,
diones and phenols.
When BaP adsorbed on CAP is added to the whole animal, the relative
amount of metabolism does not change compared to CAP alone at 180
minutes. There is an increase in the relative amount of metabolite
in the blood and a decrease in the lung while the BaP content does
not change significantly. There is a corresponding decrease in the
9,10-diol and a large increase in the nonextractables. A significant
relative increase of metabolite in the washout and relative decreases
of metabolite in trachea bronchi and macrophage are observed for BaP
adsorbed on CAP compared to CAP alone. This is consistent with
decreases in the amounts of the 7,8-and 9,10-diols in macrophages and
diones in the trachea bronchi with corresponding increases of BaP in
both tissues. Amounts of phenols increased in the trachea bronchi.
No such metabolite profile changes are present in the washout data.
These data suggest that BaP adsorbed on CAP does not change signifi-
cantly the relative amounts of metabolite and BaP in tissues at 180
minutes. However, the metabolite distribution has changed in that
there is a decrease in the diol formation and an increase of non-
extractables which suggests that BaP and CAP together act differently
than when administered separately as a pretreatment.
Summary
CAP acts as an enzyme inducer to increase the rate of metabolism of
BaP. BaP adsorbed on CAP also acts like an enzyme inducer by increasing
the rate of metabolism, but the rate of metabolism is not the sum of the
two individual rates. This indicates that the BaP is not leached
73
-------
readily from the CAP, i.e. BaP is not available for enzyme induction,
Overall CAP or BaP (results, Section Idj^by itself increase the diol
formation in the tissues with smaller increases or decreases in non-
extractables. Together BaP and CAP decrease the diol formation and
increase the nonextractables in the tissues.
74
-------
TABLE 23.
INFLUENCE OF PARTICULATE PRETREATMENT ON BaP METABOLISM
RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animal s
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S. E. )
Metabolite pattern in
blood (% ± S.E.)b
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
Monohydroxylated
Diones
Nonextractable
BaP
9
256
6
15
3
9
10
54
.6
.4
.3
.7
.6
.4
±37
X
- 0.
+
- 4.
- 0.
± 1.
±1.
- 5.
c
H
9d
H
od
6d
lc
8e
4
CAP
BaP a
5
830
18
32
0
3
5
39
.2
.6
.9
.4
.5
.4
- 100
j-
- 5.
+
- 4.
±0.
±0.
+- 1.
- 8.
6
3
5
6
8
0
(BaP 4
• CAP)n
BaP a
5
1093
11.
25.
4.
5.
7.
45.
2
9
9
7
0
2
- 153
+
- 3.4
4.
- 2.4
±2.5
± 0.9e
± 1.2
- 4.3
Columns 1 & 3 compared to Column 2. All metabolites separated by tic.
a!0 mg/kg, once a week x 5, BaP and/or CAP.
Metabolite pattern values expressed as percent of total rate of
appearance of metabolites in blood - S.E.
CP = 0.01
dP = 0.05
eP = 0.10 (Student-Newman-Keuls Test)
75
-------
TABLE 24. '
COMPARISON OF HPLC AND .tic DATA
Pretreatment
IPL
No. of Animals
Appearance of
all metabolites
in blood at
60 minutes +
(ng/g lung - S.E.)
Metabolite Pattern
(« - S.E.)W
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-,l ,6-6,12-quinone
4,5-epoxide^
9 & 6-OH
3 & 7-OH
nonextractable
S metabolite
HPLC
877 +-
27.5 -
2.8 -
1.8 -
1.3 -
5.3 -
2.5 -
2.5 -
2.4 -
43.8 -
9.6 -
BaP
5
107
J
(
4.6 :
i
0.2 j
a
0.3 i
0.0 |
0.5 i
1.1
0.4 1
0.2
5.5 v '
1.1 i
tic
967 - 85
30.1 - 3.8
0.8 - 0.5
16.0 - 3.6
5.2 - 1.2
3.9 - 0.4
43.9 - 6.0
w
Metabolite pattern value expressed as % of appearance of all metabolites
in blood at 60 minutes - standard error. :
counts appear under this peak.
76
-------
TABLE 25.
COMPARISON OF HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
(BaP + CAP)
BaP 1!
5
Appearance of
all metabolites
in blood at
60 minutes +
(ng/g lung - S.E.)
Metabolite Pattern
(°l - S F ^W
\ h O.L.y
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-,l,6-,6,12-quinone
4,5-epoxide^
9 & 6-OH
3 & 7-OH
nonextractable
S metabolite
HPLC
1152 + 214
26.0 - 5.4
2.5 - 0.5
3.1 - 0.4
1.3 - 0.3
5.1 - 0.9
3.6 - 0.6
3.1 - 0.4
3.4 - 0.6
43.5 - 6.4
8.3 - 2.0
tic
1116 - 213
28.9 - 4.6
4.4 - 1.9
11.3 - 3.2
6.3 - 1.5
6.0 - 1.1
43.0 - 6.1
wMetabolite pattern value expressed as % of appearance of all metabolites
in blood at 60 minutes - standard error.
yi
- counts appear under this peak.
77
-------
TABLE 26.
INFLUENCE OF PARTICULAR PRETREATMENT
OF TOTAL BaP AND TOTAL METABOLITE- REMAINING IN EACH TISSUE
AT 180 MINUTFSi't S.E.
Pretreatment
IPL
No. of Animals
% Unmetabolized BaP
% of Total Compound as
Metabolite in Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound as
BaP in Tissue
Blood
TB
MAC
WO
Lung
BaP
3
65.3 - 4.3
15.5 - 5.2
3.7 - 1.2
0.3 - 0.1
3.3 - 2.3
12.0 - 1.9
12.7 - 1.4
10.4 - 2.7
8.7 - 7.4
2.5 - 1.1
31.0 - 3.7
CAPIT
BaP
1
20.7
24.0
8.3
5.3
2.1
39.6
3.6
2.4
1.5
0.6
12.5
(CAP + BAP)n
BaP
2
21.3 - 2.2
31.9 - 3.5
3.5 - 2.0
0.9 - 0.2
11.8 - 4.5
30.7 - 0.2
1.6 - 0.6
2.1 - 0.1
2.3 - 0.0
2.4 - 0.3
12.9 - 0.8
78
-------
TABLE 27.
% DISTRIBUTION PATTERN OF BaP + METABOLITES IN EACH TISSUE
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxyl ated
diones
nonextractable
BaP
LUNGX
None
BaP
1.2-0.1
2.9-1.5
0.9-0.4
10.1-0,6
1.1-0.3
5.3-0.9
78.5-2.7
CAPn
BaP
7.7
20.5
0.8
8.3
10.2
18.3
34.2
(CAP+BaP)IT
BaP
7.2^2.2
7.8-2.9
1.7-0.7
5.7-1.0
4.0-0.3
32.0-5.8
41.5-1.3
y +
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals 3,1 & 2 respectively.
(continued)
79
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 27.
(continued)
180 BLOODX
None
BaP
2.8-0.5
6.5-3.8
1.2-0.7
2.5-0.9
1.3-0.1
18.6-2.5
67.0^6.5
CAPn
BaP
19.4
44.1
1.5
1.9
4.4
9.1
19.8
(CAP+BaP)n
BaP
7.0-0.7
20.8^6.7
4.4-3.9
3.0-0.0
1.6-0.1
55.1-15.1
8.0-3.7
xBased on % total activity at 180 minutes in each tissue - S.E.
Number of animals 3,1 & 2 respectively.
(continued)
80
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxyl ated
diones
nonextractable
BaP
TABLE 27.
(continued)
MACROPHAGEX
None
BaP
1.4-0.4
0.9^0.2
0.2-0.2
0.9-0.3
0.5-0.2
1.9-1.0
94.2-2.2
CAPn
BaP
35.6
14.1
1.8
2.3
10.7
3.5
32.1
(CAP+BaP)IT
BaP
3.6-1.5
3.0-1.3
0.6-0.5
3.7-1.1
3.7-0.6
5.4-0.4
81.9-4.8
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals 3,1 & 2 respectively.
(continued)
81
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 27.
(continued)
WASHOUTX
None
BaP
5.0-1.8
10.8-8.3
0.8-0.6
2.7-2.0
2.1-1.3
17.5-6.6
61.0-7.7
CAP,,
BaP
11.2
13.7
0.5
1.2
8.2
30.8
34.3
(CAP+BaP)n
BaP
10.4-3.0
16.9-3.7
1.3-0.7
4.4-0.4
11.0-6.5
27.4-3.2
28.6-10.2
xBased on % total activity at 180 minutes in each tissue - S.E.
Number of animals 3,1 & 2 respectively.
(continued)
82
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 27.
(continued)
TRACHEA BRONCHI
None
BaP
0.8-0.2
2.2-1.2
0.9-0.5
3.4-1.8
2.1-1.4
12.5-4.1
79.4-7.4
X
CAPn
BaP
4.1
4.0
0.0
6.5
38.1
15.2
32.1
(CAP+BaP)n
BaP
3.9-0.1
4.0-1.5
1.0-0.4
20.4-16.6
2.9-2.9
13.7-1.7
54.1-13.8
X +
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals 3,1 & 2 respectively.
83
-------
d. Influence of Crude Air Particulate on BaP Metabolism
1) Rate of Metabolism
CAP pretreatment appears to increase enzyme activity as indicated
by the rate of metabolism (Table 28). The CAP administered con-
currently with BaP on IPL, on the other hand, appears to influence
metabolism in the same manner as described previously, i.e., increased
rate of action of macrophages or the slow physical release of BaP-
The distribution indicates that CAP pretreatment increases the amount
of nonextractable and the 7,8- and 9,10-diol formation versus the
control. On the other hand, CAP with BaP on the IPL causes a signif-
icant decrease in the 9,10-diol and an increase in the diones and
monohydroxylated compounds. This could reflect a partial decrease in
epoxide hydrase activity.
These data suggest that CAP is affecting BaP metabolism by two
different mechanisms: One mechanism appears to be a long-term effect,
i.e., increasing total metabolic activity, and the other is a short-term
effect with a decrease in total metabolic activity which overrides the
effects of pretreatment.
The HPLC supports the tic data (Table 29). It should be noted
the 7,8-dihydrodiol on tic, 23.6%, actually consists of the 7,8-dihy-
drodiol plus the S metabolite (24.5%). All the other values of HPLC
agree extremely well with tic (25,55).
2) Distribution of BaP and Its Metabolites in Tissues at
180 Minutes
CAP given as a pretreatment (based on one experiment at 180
minutes) to the whole animal increases the amount of metabolite found
in tissues at 180 minutes (Table 30 & 31). There is a corresponding
relative increase of metabolite found in the blood and lung and a
relative decrease in BaP. This is reflected in an increase in the 9,10-
and 7,8-diols and a decrease in the BaP content in both tissues. The
nonextractables increase in the lung and decrease in the blood compared
to the control. There are some small relative increases of metabolite
in the trachea bronchi and macrophage and corresponding decreases of
BaP in these tissues, while there are slight relative decreases in
metabolite and BaP in washout compared to the control. These data are
consistent with significant increases of 7,8- and 9,10-diols and
quinones in both the macrophage and washout, increases of the diones in
the trachea bronchi and a significant increase of the nonextractables in
the washout. These data suggest that CAP given as a pretreatment acts
as an enzyme inducer; an increase in the rate of metabolism of BaP is
reflected in an increase of diol formation in the tissues of the lung
and smaller increases in the nonextractables, diones, and phenols.
84
-------
When CAP is added to the whole animal followed by BaP adsorbed
on CAP on the IPL, the relative amount of metabolism decreases com-
pared to its control at 180 minutes (Table 30,31). There is a decrease
in relative amount of metabolite in the blood and lung while there
is an increase in BaP in the lung. There is a corresponding increase
in nonextractables in the blood with decreases of 9,10-diol for blood
and lung. A significant relative increase of BaP in the macrophage is
observed for BaPjj followed by BaP + CAP on IPL compared to its control.
There are no such changes observed for washout or trachea bronchi.
These latter observations are consistent with a decrease in the 7,8-
and 9,10-diols and diones in the macrophage while the washout shows
an increase in the 7,8-dihydrodiol and nonextractables and the trachea
bronchi shows a significant decrease in diones.
These data suggest that CAPjy followed by BaP adsorbed on CAP
significantly changes the relative amounts of metabolite and BaP in
tissues at 180 minutes compared to CAPjj followed by BaP on IPL. The
metabolite distribution has also changed in that there is a decrease
in the diol formation and an increase in the nonextractables which
suggests that BaP and CAP on the IPL tends to effect our type of
pretreatment.
Summary
CAP acts as an enzyme inducer to increase the rate of metabolism
of BaP. BaP adsorbed on CAP on the IPL on the other hand, appears to
inhibit the action of CAPjj pretreatment. Overall, these observations
are reflected in decreases in diol formation and increases in BaP
content. The major increase of BAP is in the macrophage which is
consistent with decrease in metabolism; the BaP adsorbed on CAP
administered to the IPL causes an increase in the rate of action of
macrophage.
85
-------
TABLE 28.
INFLUENCE OF CRUDE AIR PARTICULATE ON BaP METABOLISM
RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animal s
Total rate of appearance
of metabolites in blood
(ng/hr/g lung - S.E. )
Metabolic pattern in
blood (% - S.E.)W
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxyl ated
diones
nonextractable
BaP
9
256 - 37a
6.6 - 0.9b
15.4 - 4.0b
3.3 t 0.6b
9.7 - l.la
10.6 - 1.8C
54.4 - 5.4
PAP ^ TAP
o*\r y ^- *-»nr T-
BaP BaP +
5 5
830 - 100 143 -
18.2 - 5.6 23.6 -
32.6 - 4.3 17.0 -
0.9 - 0.5 1.9 -
3.4 - 0.6 6.5 -
5.5 - 1.6 10.4 -
39.4 - 8.0 40.6 -
z
r
CAPX
29a
7.6
7.2C
0.7
2.3
2.3
6.6
Column 1 compared to Column 2 - Column 3 compared to Column 2
Metabolite pattern values expressed as percent of total rate of
appearance of metabolite in blood - standard error.
Xl mg/kg
Z10 mg/kg, once/wk x 5
a. P = 0.01 b. P = 0.05 c. P = 0.10
(by Student-Newman-Keul s Test)
All metabolites separated by tic.
86
-------
TABLE 29.
COMPARISON OF HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
BaP + CAP
5
Appearance of
all metabolites
in blood at
60 minutes
(ng/g lung - S.E.
Metabolite pattern
("I - <; F \
\ 10 O . L • ^
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-,l ,6-,6,12-quinone
4,5-epoxide-y
9 & 6-OH
3 & 7-OH
nonextractable
S metabolite
HPLC
150-25
13.9 - 5.0
3.7 - 1.4
2.3 - 0.4
3.8 - 0.9
3.6 - 0.9
2.4 - 0.5
3.1 - 0.9
4.1 - 0.8
40.9 - 5.9
22.2 - 5.9
tic
144 + 31
16.3 - 6.0
1.6 - 0.4
23.6 - 7.8
10.0 - 2.0
5.1 - 1.7
43.4 - 6.7
wMetabolite pattern value expressed as % of appearance of all
metabolites in blood at 60 minutes - standard error.
counts appear under this peak.
87
-------
TABLE 30.
INFLUENCE OF PARTICULATE ON BaP METABOLISM ON IPL
% OF TOTAL BaP AND TOTAL METABOLITE REMAINING
IN EACH TISSUE AT 180 MINUTES - S.E.
Pretreatment
IPL
No. of Animal s
% Unmetabolized BaP
% of Total Compound as
Metabolite in Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound as
BaP in Tissue
Blood
TB
MAC
WO
Lung
BaP
3
65.
15.
3.
0.
3.
12.
12.
10.
8.
2.
30.
3
5
7
3
3
0
7
5
7
5
9
±4.
±5.
±1.
±0.
±2.
± 1.
±1.
±2.
±7.
± 1.
±3.
3
2
2
1
3
9
4
7
4
1
7
CAPn
BaP
1
20.
24.
8.
5.
2.
39.
3.
2.
1.
0.
12.
7
0
3
3
1
6
6
4
5
6
5
BaP+CAP
3
52.6 ± 11.2
13.8 ± 2.7
4.3 ± 1.4
4.9 ± 0.7
3.9 ± 0.6
20.5 ± 3.6
1.4 ± 0.5
4.3 ± 1.6
29.5 - 6.5
0.4 ± 0.1
17.1 ± 3.3
88
-------
TABLE 31.
% DISTRIBUTION PATTERN OF BaP + METABOLITES IN EACH TISSUE
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxyl a ted
diones
nonextractable
BaP
LUNGX
None
BaP
1.2-0.1
2.9-1.5
0.9-0.4
10.1-0.6
1.1-0.3
5.3-0.9
78,5-2.7
CAP
BaP
7.7
20.5
0.8
8.3
10.2
18.3
34.2
CAP^
BaP + CAP
5.9-0.6
6.0-2.0
2.4-0.8
4.4-1.5
2.1-1.3
13.6-6.0
66.6-4.2
y +
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals per experiment 3,1 & 3 respectively.
(continued)
89
-------
TABLE 31.
(continued)
180 BLOODX
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4s5-dihydrodiol
monohydroxylated
diones
nonextrac table
BaP
None
BaP
2.8-0.5
6.5-3.8
1.2-0.7
2.5-0.9
1.3-0.1
18.6-2.5
67.0-6.5
CAP
BaP
19.4
44.1
1.5
1.9
4.4
9.1
19.8
CAPn
BaP + CAP
8.4-2.3
14.3-9.8
1.7-0.5
8.9-4.3
4.5-1.4
40.5-17.9
21.6-8.5
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals per experiment 3,1 & 3 respectively.
(continued)
90
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 31 .
(continued)
MACROPHAGEX
None
BaP
1.4-0.4
0.9^0.2
0.2-0.2
0.9-0.3
0.5-0.2
1.9-1.0
94.2-2.2
CAP
BaP
35.6
14.1
1.8
2.3
10.7
3.5
32.1
CAPJT
BaP + CAP
2.2-0.6
0.4^0.1
0.0-0.0
0.4^0.2
2.2-1.1
2.0-0.6
92.7-2.5
X +
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals per experiment 3,1 & 3 respectively.
(continued)
91
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 31.
(continued)
WASHOUT*
None
BaP
5.0-1.8
10.8-8.3
0.8-0.6
2.7-2.0
2.1-1.3
17.5-6.6
61.0-7.7
CAP
BaP
11.2
13.7
0.5
1.2
8.2
30.8
34.3
CAPIT
BaP + CAP
23.4-7.0
12.1-4.6
1.3-0.2
2.4-0.5
4.1-0.5
37.4^9.2
19.4-1.8
(Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals per experiment 3,1 & 3 respectively.
(continued)
92
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 31.
(continued)
TRACHEA BRONCHI
None
BaP
0.8-0.2
2.2-1.2
0.9-0.5
3.4-1.8
2.1-1.4
12.5-4.1
79.4-7.4
X
CAP
BaP
4.1
4.0
0.0
6.5
38.1
15.2
32.1
CAPn
BaP + CAP
6.0-1.4
6.1-3.3
0.6-0.1
4.2-2.3
3.3-2.3
11.9-2.6
67.9-4.3
X +
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals per experiment 3,1 & 3 respectively.
93
-------
3. Effects of S02
a. Influence of S02 Pretreatment on BaP Metabolism
1) Rate of Metabolism
S02 pretreatment significantly increases the rate of metabolism
of BaP when compared to its control (Table 32a & b); but this increase
is significantly smaller when compared to BaP pretreatment, a well
characterized MFO (mixed function oxidase) enzyme inducer. S02,
therefore, acts as a biochemical agent which causes biochemical changes
in the lung due to irritation of S02 (15,26,42).
The distribution data do not show any marked changes when S02
pretreatment is compared to its control. There is a slight increase
in the nonextractables and a slight decrease in monohydroxylated com-
pounds and a larger decrease in dione formation. However, BaP pretreat-
ment, on the other hand, shows a marked increase in the 9,10-dihydrodiol
and a smaller decrease in monohydroxylated and diones when compared to
S02 pretreatment or its control. This indicates that the S02 pretreat-
ment does not have a marked effect on the metabolic pathways, i.e.
epoxidation, hydroxylation and/or isomerization and conjugation
(37,39,72,73).
In order to validate the tic data, the HPLC data for S02 pretreat-
ment is compared to the tic data (Table 33) which not only supports
but complements the data in Table 32a. The rates of formation, the
4,5-dihydrodiols, the nonextractables and the hydroxylated metabolites
are very comparable. There are some differences, however, which are
not totally unexpected considering the differences in the techniques.
There is an increase in the quinones and a small decrease in 9,10-di-
hydrodiol for the HPLC data when compared to the tic data. The
7,9-dihydrodiol and the S metabolite (ninhydrin positive and fluo-
rescent which appears after BaP), on HPLC, chromatograph together on
tic and show some differences.
The HPLC data for the control are compared to the tic data
(Table 34) using four samples. These data show essentially no differ-
ences between the tic and HPLC methods (25,55).
2) Distribution of BaP and Its Metabolites in Tissue at
180 Minutes
At 180 minutes, more BaP has been metabolized in the IPL under
BaPjj pretreatment conditions compared to S02 or no pretreatment sit-
uation (Table 35). These results are consistent with rate of appear-
ance of metabolites in blood (Table 32a). When S02 is given a
pretreatment there is a significant increase in the amount of metabo-
lite in lung and a decrease in the amount of BaP in trachea bronchi and
94
-------
macrophage (Table 36, 37). This is reflected in an increase in the
nonextractables and a decrease in the monohydroxylated compounds in
the lung. The washout data shows a decrease in the 9,10-diol and
nonextractables, trachea bronchi show an increase in the nonextract-
ables and blood and macrophage do not show any significant changes in
metabolite pattern.
95
-------
TABLE 32a
INFLUENCE OF S02 PRETREATMENT ON BaP METABOLISM
RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animals
Total rate of appearance
of metabolites in blood
None
BaP
9
256-38
(so2)|T
BaP
5
514-95d
BaP
5
1290-114d'e
(ng/hr/g lung - S.E. )
Metabolic pattern in
blood (% - S.E.)C
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
Monohydroxylated
Diones
Nonextractables
6.6-0.9
15.4-4.0
4-
3.3-0.6
9.7-1.1
10.6-1.8
54.4-5.4
6.0-1.0
15.3-6.6
4. f
1.2-0.41
7.9-1.7
3.8-0.86
65.7-0.4
5.4-1.8
32.8-3.8f
4- f
1.8-0.51
5.9-1.0
2.8-Q.96
51.8-2.8
All metabolites separated by tic. All columns compared to each other.
1.6 - 0.4 ppm, 45 minutes.
10 mg/kg once/week x 5.
Metabolite pattern values expressed as percent of total rate of
appearance of metabolite in blood - S.E.
dP = 0.05
P - 0.01
fP = 0.1
(Statistics performed by Student-Newman-Keuls Test)
96
-------
TABLE 32b
*ETREATMEN1
RATE AND PATTERN OF METABOLISM IN THE BLOOD
INFLUENCE OF S02 PRETREATMENT ON BaP METABOLISM
Pretreatment
IPL
No. of Animal s
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S.E. )
None
BaP
4
334-40
(S02)a
BaP
5
547-69c
Metabolic,pattern in
blood (% - S.E.)b
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
1 ,6-6, 12-3, 6-quinone
4,5-epoxidec'
9,6-OH
7-OH
3-OH
S Metabolite
Nonextractable
20.1-6.4
2.8*0.4
2.7*0.6
1.4*0.6
2.3*0.6
2.3*1.0
2.1-0.9
2.6±0.3e
9.4*3.8
54.3*7.7
10.4*4.3
2.5*0.6
4.0*0.6
2.3*0.3
5.0*1.8
2.5*0.5
2.6-0.7
2.2*0.6f
1.6*0.5
7.7*2.4
59.0*4.7
All metabolites separated by HPLC. Columns compared to each other.
al,6 - 0.4 ppm, 45 minutes.
Metabolite pattern values expressed as percent of total rate of
appearance of metabolites in blood * S.E.
CP = 0.5 (Student-Newman-Keuls Test)
14- counts appear under this peak.
e7-OH and 3-OH collected together.
f7-OH & 3-OH combined 1.9 - 0.4.
97
-------
TABLE 33.
COMPARISON HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
so2
BaP
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S. E.)
Metabolic pattern in
blood (% * S.E.)a
HPLC
547-69
tic
514-95
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4 ,5-quinone
1 ,6-6, 12-3, 6-quinone
4,5-epoxide
9, 6- OH
7-OH
3-OH
S Metabolite
Nonextractable
10.4-4.3 15.3-6.6
2.5-0.6 1.2-0.3
4.0-0.6 6.0-1.0
2.3-0.3
5.0-1.8
2.5-0.5
-L
2.6-0.7
2.2-0.6
1.6-0.5
— 3.8-0.8
— 7.9-1.6
7.7-2.4
59.0-4.7 65.7-0.4
Metabolite pattern values expressed as percent of total rate of
appearance of metabolites in blood - S.E.
14C counts appear under peak.
98
-------
TABLE 34.
COMPARISON OF HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
BaP
4
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S.E.)
Metabolic pattern in
blood (% - S.E.)a
HPLC
334-40
tic
364-45
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
1 ,6-6, 12-3, 6-quinone
4,5-epoxide
9,6-OH
7-OH
3-OH
S Metabolite
Nonextractable
20.1-6.4 18.3-7.
2.8-0.4 2.2-1.
2.7-0.6 10.3-2.
1.4-0.6
2.3-0.6
2.3-1.0
_i_
2.1-0.9
2.6-0.3C
? fi+n
o . o u .
6 3+2
U • O L. •
9.4-3.8
54.3-7.7 59.2-9.
5
0
7
8
7
9
^Metabolite pattern values expressed+as percent of total rate of
appearance of metabolites in blood - S.E.
D14C counts appear under peak.
:7-OH and 3-OH collected together.
99
-------
TABLE 35.
INFLUENCE OF S02 PRETREATMENT ON BaP METABOLISM*
% OF TOTAL BaP AND TOTAL METABOLITE REMAINING IN
EACH TISSUE AT 180 MINUTES - S.E.
Pretreatment
IPL
No. of Animals
% Unmetabolized BaP
% of Total Compound as
Metabolite in Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound as
BaP in Tissue
Blood
TB
MAC
WO
Lung
BaP
3
65.3 -
15.5 -
3.7 +
0.3 +
3.3 +
12.0 -
12.7 +
10.4 -
8.7 +
2.5 +
30.9 -
4.3
5.2
1.2
0.1
2.3
1.9
1.4
2.7
7.4
1.1
3.6
so2*
BaP
3
46.9 + 5.2
16.2 + 3.0
6.2 + 1.4
0.5 - 0.1
2.9 - 0.2
26.9 + 6.0
10.5 - 3.3
2.9 + 1.4
2.0 + 0.4
3.0 - 0.3
28.6 - 3.4
BaPIT
BaP
3
21.9 - 3.1
41.5 - 7.5
1.3 - 0.5
0.9 - 0.2
6.3 - 0.5
28.0 - 1.4
1.9 - 0.2
1.5 - 0.5
4.7 - 0.6
2.4 - 0.6
11.5 - 3.0
*tlc data
100
-------
TABLE 36.
% DISTRIBUTION PATTERN OF BaP + METABOLITES IN EACH TISSUE11
LUNGX
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
None
BaP
1.2-0.1
2.9-1.5
0.9-0.4
10.1-0.6
1.1-0.3
5.3-0.9
78.5-2.7
S09*
2
BaP
3.8-0.6
2.2-0.4
0.2-0.1
2.2-0.2
2.4-0.3
10.0-0.7
79.2-1 .9
BaPTT
IT
BaP
5.4-0.7
2.7-0.6
0.9-0.3
3.5-1.4
3.7-0.7
43.9-4.6
39.8-7.4
*S02 - 120 minutes
BaP
y +
Based on % total activity at 180 minutes in each tissue - S.E.
Hhere are 3 animals each for controls, S02, and BaPIT - tic data.
(continued)
101
-------
TABLE 36.
(continued)
180 BLOODX
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxyl ated
diones
nonextractable
BaP
None
BaP
2.8-0.5
6.5^3.8
4.
1.2-0.7
2.5^0.9
4.
1.3-0.1
18.6-2.5
67.0-6.5
SO *
c_
BaP
1.9-0.3
2.4-0.4
4.
0.3-0.1
2.2-0.3
4.
3.3-0.9
20.8-2.6
69.1-2.8
BaPTT
1 1
BaP
6.4-1.4
17.4-2.5
4.
1.9-1.2
3.3-0.6
J_
1.9-0.4
61.5-2.5
7.6-1.4
*so
iU2 = 120 minutes
BaP
xBased on % total activity at 180 minutes in each tissue - S.E.
There are 3 animals each for controls, SO^, and BaPJT - tic data.
(continued)
102
-------
TABLE 36.
(continued)
MACROPHAGEX
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
None
BaP
1.4-0.4
0.9-0.2
0.2-0.2
0.9-0.3
+
0.5-0.2
1.9-1.0
94.2-2.2
so;
BaP
1.2-0.2
0.7-0.1
0.1-0.0
2.8^0.6
4.
0.8-0.2
1.5-0.2
93.0-1.1
BaPIT
BaP
1.4-0.3
0.9-0.2
0.1-0.0
1.3-0.6
+
2.4-1.0
3.5-0.9
90.3-2.5
= 120 minutes
BaP
X +
Based on % total activity at 180 minutes in each tissue - S.E.
There are 3 animals each for controls, SO^, and BaPjT - tic data.
(continued)
103
-------
TABLE 36.
(continued)
WASHOUTX
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxyl ated
diones
nonextractable
BaP
None
BaP
5.0-1.8
10.8-8.3
0.8-0.6
2.7-2.0
2.1-1.3
17.5-6.6
61.0-7.7
so2*
BaP
2.0-0.5
2.3-0.7
0.5-0.1
5.5-0.3
3.3-1.9
6.9-2.3
78.4-2.9
BaPIT
BaP
5.5-1.8
7.1-1.2
0.6-0.4
3.8-1.1
3.8-1.3
39.6-4.6
39.6-2.2
*so
2 = 120 minutes
BaP
x +
Based on % total activity at 180 minutes in each tissue - S.E.
There are 3 animals each for controls, SO,,, and BaPIT tic data.
(continued)
104
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 36.
(continued)
TRACHEA BRONCHI
None
BaP
0.8-0.2
2.2-1.2
0.9-0.5
3.4-1.8
2.1-1.4
12.5-4.1
79.4-7.4
X
so2*
BaP
3.4-1.5
1.6-0.3
0.2-0.1
1.7-0.2
5.7-3.2
27.0-6.2
60.4-6.1
BaPn
BaP
4.4-0.7
3.4-0.8
0.5-0.2
1.4-0.7
3.2-2.6
24.8-11.5
62.2-10.1
= 120 minutes
BaP
x +
Based on % total activity at 180 minutes in each tissue - S.E.
There are 3 animals each for controls, SO,,, and BaPjy - tic data.
105
-------
BaPjj pretreatment shows a large increase in metabolite in the
blood, a corresponding decrease of BaP in the blood and lung when
compared to S02 and/or control. There is a comparable increase of
metabolite in the lung as in the case for S02 pretreatment. This is
reflected in an increase in the nonextractables in the lung, washout,
and blood, and an increase in 9,10-diol in the blood as shown in
Table 32a for BaPjy pretreatment compared to S02 pretreatment and/or
control. An increase in the nonextractables in the trachea bronchi
is present for both S02 and BaP pretreatment.
These data suggest that on a percentage basis more metabolite
is found in the IPL, i.e. lung, washout, and blood, after BaPjj pre-
treatments than S02 pretreatments and controls at 180 minutes. This
is reflected in a general increase in the nonextractables in lung,
washout and blood.
Summary
These data indicate that initially S02 pretreatment increases
the rate of metabolism compared to control but does not significantly
change the metabolic pattern. The S02 pretreatment data, however,
are very different from that obtained for an enzyme inducer which
affects not only the rate but also the pattern. This difference in
rate between BaPjj and S02 and control is reflected in a large increase
in the 9,10-diol and a slight decrease in the nonextractables.
After 180 minutes, similar results are obtained in that the
amount of BaP unmetabolized in each case is consistent with rate of
metabolism. In general, there are some small increases in the amount
of nonextractables for S02 pretreatments compared to control while
there are some rather large increases in the nonextractables from BaPjj
pretreatments compared to S02 and control. These data again suggest
that S02 pretreatments show an increase in the rate of metabolism but
does not significantly affect the pattern. S02, therefore, does not
appear to affect BaP metabolism by enzymatic induction.
106
-------
b. Influence of S02 Administered to IPL on BaP Metabolism
1) Rate of Metabolism
S02 administered to the IPL significantly increases the rate of
metabolism of BaP compared to its control (Tables 37,40). This suggests,
in the same manner described previously, that S02 acts as a biochemical
agent which causes an increase in the enzyme activity or as a physical
agent which causes biochemical changes in the lung (26,42). However,
CAP administered concurrently with BaP and S02 to the IPL, appears to
significantly decrease the rate of metabolism of BaP when compared to
its appropriate control. A factor to consider is that BaP may not be
leached readily from the CAP and therefore, is not available to be
metabolized.
The metabolite pattern does not show any marked changes when the
S02 treatment is compared to its control. There are small increases in
the 9,10-dihydrodiol and nonextractables and small decreases in mono-
hydroxylated and dione formation. When CAP is administered concurrently
with BaP and S02 to the IPL there is a decrease in the nonextractables
and small increases or no change in the 9,10- and 7,8-dihydrodiols
(Tables 37,40). This indicates that S02 administered in vitro on the
IPL does not have major effects on the metabolic pattern.
In order to validate the tic data, the HPLC data for S02 and
BaP on the IPL, as well as S02, CAP and BaP on the IPL are compared
to tic (Tables 38,39). The rates, nonextractables, the monohydroxylated
and 4,5- and 9,10-dihydrodiols are very comparable. The diones in
Table 38 are comparable while the diones in Table 39 show an increase
for HPLC data when compared to the tic data. In both Tables 38 and
39 the 7,8-dihydrodiol from the tic data is very comparable to the
7,8-dihydrodiol plus the S metabolite (appears after BaP on chromato-
gram and is fluorescent and ninhydrin positive) for HPLC data (25,55).
2) Distribution of BaP and Its Metabolites in Tissue at 180
Minutes
At 180 minutes, there is a small decrease in the (Tables 40,41)
amount of unmetabolized BaP compared to control. This is consistent
with small increases of metabolite in blood and lung and BaP in the
blood with corresponding decreases of BaP in trachea bronchi and lung.
These data are consistent with small increases in the 7,8- and 9,10-
diols in lung, blood washout, macrophage, and trachea bronchi and
other minor changes in monohydroxylated diones and nonextractables.
Therefore, as similarly stated in Section b,l), SO with BaP on IPL
does not have major effects on the metabolic pattern.
107
-------
When CAP and S02 are administered together with BaP on the IPL,
there appears to be a dramatic increase in metabolism of BaP after
180 minutes compared to S02 and BaP alone (Tables 40,41). There also
appears a change in the rate of metabolism of BaP due to CAP plus
S02 as seen from the results in Tables 37 and 41. Initially the BaP
may not be leached readily from CAP and therefore, is not available
to be metabolized. However, after a period of time, the BaP is probably
leached from the CAP at a faster rate. By this time the S02 has had
an effect on the lung such as to cause cellular injury and/or a change
in normal enzyme functions. These changes result in an increase in the
amount of BaP metabolized.
These observations and assumptions are reflected in large increases
of metabolite in blood, trachea bronchi and lung and large decreases of
BaP in blood and lung compared to S02 and BaP alone. These data are
consistent with relative increases in diol and nonextractables in blood
and washout and relative increases in diones and nonextractables in
the trachea bronchi. No such changes are seen for the lung and macro-
phage. This last statement is not consistent with the data obtained
for the lung as seen in Table 41.
All of these data suggest that CAP and S02 administered together
with BaP on the IPL causes an increase in the rate of metabolism after
an equilibrium period compared to control. This increase is reflected
in more metabolite in blood, trachea bronchi, and lung at 180 minutes
with most of the metabolite increase attributed to the 7,8- and/or
9,10-diol and nonextractable formation.
Summary
These data indicate that initially S02 on the IPL increases the
rate of metabolism of BaP compared to control but does not signifi-
cantly change the metabolite pattern. When CAP and S02 are administered
to the IPL, the rate of metabolism of BaP is significantly decreased
compared to its appropriate control. This could be due to the fact that
BaP is not leached readily from the CAP. The CAP and S02 together do,
however, cause small changes in the metabolic pattern compared to the
control with increases in the 7,8- and 9,10-diol and a decrease in
nonextractables.
At 180 minutes, the amount of unmetabolized BaP left for BaP and
BaP plus S02 are consistent with rate of metabolism. The S02 does not
appear to have major effects on the metabolic pattern. CAP plus SOz
administered together with BaP, on the other hand, causes a signifi-
cant increase in the rate of metabolism of BaP after an equilibration
period. This can be due to the combination of leaching of BaP from
CAP at a faster rate and changes in normal enzyme functions and/or
cytopathological effects due to the S02. This increase in metabolism is
attributed to the 7,8- and/or 9,10-diol and nonextractable formation.
Under these conditions, therefore, S02 may cause an increase in the
enzyme activity.
108
-------
TABLE 37.
INFLUENCE OF S02 ADMINISTERED TO IPL ON BaP METABOLISM
RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animals
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S.E. )
Metabolic pattern in
blood (% - S.E.)C
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
Monohydroxylated
Diones
Nonextractables
None
BaP
9
256-38d
6.6^0.9
15.4-4.0
3.3-0.6
9.7-l.ld
10.6-1.8
54.4-5.4e
None
BaP+S02a
5
551-77
6.0-0.7
20.0-2.1
2.0-0.6
4.2-0.6
7.9-2.6
59.9-3.4
None
BaP+CAP+S02b
7
177-48d
n.o-i.6f
26.7-2.9e
1.7-0.7
6.3-1.1
6.9-2.0
42.4-2.6d
All metabolites separated by tic. Columns 1 & 3 compared to Column 2.
a2.2 - 0.1 ppm S02
b!0 mg/kg CAP, 2.1 - 0.1 ppm S02
cMetabolite pattern values expressed+as percent of total rate of
appearance of metabolites in blood - S.E.
dP = 0.01
eP = 0.1
fP = 0.05
(Statistics performed by Student-Newman-Keuls Test)
109
-------
TABLE 38.
COMPARISON HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S.E. )
Metabolic pattern in
blood (% - S.E. )a
9,10-dihydrodiol
4,5-dihydrodiol
7 ,8-dihydrodiol
4,5-quinone
1 ,6-,6,12-,3,6-quinones
4,5-epoxide
9-,6-OH
7-OH
3-OH
S Metabolite
Nonextractable
None
BaP+S02
5
HPLC tic
588-60 551-77
18.9-2.1 20.0-2.
2.5-0.6 2.0-0.
2.0-0.4
2.0-0.3
3.3-1.1
4.4-0.5
2.7-0.4
1.7^0.5
1.4-0.4
6.0-0.4
6.0-0.
7.9-2.
40 n
. c.-(j.
55.1-4.5 59.9-3.
1
6
7
6
6
4
Metabolite pattern values expressed+as percent of total rate of
appearance of metabolites in blood S.E.
counts appear under peak.
110
-------
TABLE 39.
COMPARISON HPLC AND tic DATA
Pretreatment
IPL
No. of Animals
None
BaP+S02
7
+CAP
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S.E.)
Metabolic pattern in
blood (% - S.E.)a
HPLC
191-53
tic
177-47
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
1 ,6-,6,12-,3,6-quinones
4,5 epoxide
9-,6-OH
7-OH
/ \J i l
3-OH
S Metabol ite
Nonextractable
17.4-3.5 26.7-2
3.5-0.6 1.7-0
5.7-0.7 11.0-1
4.3^1.9
5.0-0.9
5.9-0.6
,
2.5-0.4
3.0-0.6
2.8-0.8
6.9-2
6 3-1
5.9-1.2
43.9-4.0 42.4-2
.9
.7
.6
.0
.1
.6
Metabolite pattern values expressed+as percent of total
appearance of metabolites in blood - S.E.
rate of
counts appear under peak.
Ill
-------
TABLE 40.
INFLUENCE OF S02 ADMINISTERED TO IPL ON BaP METABOLISM RATE
AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animals
Total rate of appearance
of metabol ites+in blood
(ng/hr/g lung - S.E. )
Metabolic pattern in
blood (% - S.E.)C
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
1 ,6-6, 12-3, 6-quinone
4,5-epoxide^
9,6-OH
7-OH
3-OH
S Metabolite
Nonextractable
None
BaP
4
334-406
20.1-6.4
2.8±0.4
2.7-0.6
1.4-0.6
2.3-0.6
2.3-1.0f
2.1-0.9
2.6-0.3f'h
9.4-3.8
54.3-7.7
None
BaP+SO?a
5 ^
588-60
18.9-2.1
2.5-0.6
2.0-0.4
2.0-0.3
3.3-1.1
4.4-0.5
2.7-0.4
1.7-0.51
1.4-0.41
6.0-0.4
55.1-4.5
None .
BaP+CAP+SO°
7 ^
191 -53d
17.4-3.5
3.5-0.6
5.7-0.7d
4.3-1.9
5.1-0.9
5.9-0.6
2.5-0.4
3.0-0.6
2.8-0.8
5.9-1.1
43.9-4.0f
All metabolites separated by HPLC. Columns 1 & 3 compared to Column 2.
a2.2 - 0.1 ppm S02
b!0 mg/kg CAP, 2.1 - 0.1 ppm S0?
Q ^
Metabolite pattern values expressed as percent of total rate of
appearance of metabolites in blood - S.E.
dP - 0.01
eP = 0.05
fP = 0.1 (Student-Newman-Keuls Test)
914r counts appear under this peak.
i L»
n7-OH and 3-OH collected together.
VOH and 3-OH combined 1.6-0.3
112
-------
TABLE 41 .
INFLUENCE OF S02 ADMINISTERED TO IPL ON BaP METABOLISM*
% OF TOTAL BaP AND TOTAL METABOLITE REMAINING IN EACH
TISSUE AT 180 MINUTES - S.E.
Pretreatment
IPL
Mo. of Animals
% of Unmetabolized BaP
% of Total Compound as
Metabolite in Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound as
BaP in Tissue
Blood
TB
MAC
WO
Lung
BaP
3
65.3 - 4.3
15.5 - 5.2
3.7 - 1.2
0.3 - 0.1 •
3.3 - 2.3
12.0 - 1.9
12.7 - 1.4
10.4 - 2.7
8.7 - 7.4
2.5 - 1.1
30.9 - 3.6
BaP+S02
3
55.2 - 4.7
17.4 - 2.4
3.3 - 1.9
0.9 - 0.1
3.8 - 0.6
19.4 - 4.8
18.8 - 8.6
2.4 - 0.6
3.6 - 1.1
3.4 - 0.8
26.9 - 0.6
BaP+S02+CAP
2
18.5 - 8.6
26.7 - 11.4
12.6 - 7.9
2.9 - 0.9
4.7 ~ 0.4
34.5 - 6.9
1.2 i 0.1
1.1 - 0.1
2.2 - 0.7
0.2 - 0.0
13.8 - 6.3
*tlc data
113
-------
TABLE 42.
DISTRIBUTION PATTERN OF BaP + METABOLITE IN EACH TISSUE
Pretreatment
IPL
7 ,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
LUNG X
None
BaP
1.2-0.1
2.9-1.5
0.9-0.4
10.1-0.6
1.1-0.3
5.3-0.9
78.5-2.7
None
BaP+S02
2.0-0.3
6.7-1.4
0.3-0.1
5.6-0.8
1.3-0.3
9.7-3.4
74.3-4.8
None
BaP+S02+CAP
3.8-0.7
3.9-0.3
0.5-0.0
5.6-2.5
3.4-0.1
9.6-2.2
73.2-5.5
X +
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,3,2 respectively - tic data.
(continued)
114
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 42.
(continued)
180 BLOODX
None
BaP
2.8-0.5
6.5-3.8
1.2-0.7
2.5-0.9
1.3-0.1
18.6-2.5
67.0-6.5
None
BaP+S02
3.1-1.4
10.2-2.7
1.0-0.6
1.8-0.1
3.4-1.8
19.4-9.4
61.1-13.7
None
BaP+S02+CAP
8.0-2.5
16.6-5.2
0.8-0.0
3.6-1.9
5.9-2.7
36.5-1.2
28.6-8.4
X +
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,3,2 respectively - tic data.
(continued)
115
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 42.
(continued)
MACROPHAGEX
None
BaP
1.4-0.4
0.9-0.2
0.2-0.2
0.9-0.3
0.5-0.2
1.9-1.0
94.2-2.2
None
BaP+S02
4.6-2.1
1.8-0.2
0.2-0.0
1.4-0.6
1.6-0.5
1.8-0.5
88.4-2.0
None
BaP+S02+CAP
8.1-2.1
0.9-0.0
0.2-0.1
0.5-0.1
4.2-1.9
1.0-0.1
85.0-0.2
xBased on % total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,3,2 respectively - tic data.
(continued)
116
-------
TABLE 42.
(continued)
WASHOUTX
Pretreatment None None None
IPL BaP BaP+S02 BAP+S02+CAP
7,8-dihydrodiol 5.0-1.8 10.5-6.1 20.0-10.1
9,10-dihydrodiol 10.8-8.3 10.0-1.6 9.6-6.0
4,5-dihydrodiol 0.8-0.6 2.1-1.5 0.9-0.5
monohydroxylated 2.7-2.0 4.5-1.5 3.1-1.8
diones 2.1-1.3 2.3-0.8 8.4-6.5
nonextractable 17.5-6.6 7.3-1.6 33.4-3.0
BaP 61.0-7.7 63.1-5.6 24.5-1.6
X
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,3,2 respectively - tic data.
(continued)
117
-------
TABLE 42.
(continued)
TRACHEA BRONCHIX
Pretreatment None None None
IPL BaP BaP+S02 BaP+S02+CAP
7,8-dihydrodiol 0.8-0.2 1.9-0.3 2.9-0.8
9,10-dihydrodiol 2.2-1.2 4.8-1.1 4.0-0.6
4,5-dihydrodiol 0.9-0.5 0.6-0.3 0.3-0.1
monohydroxylated 3.4-1.8 7.9-3.9 4.1-2.7
diones 2.1-1.4 10.4-9.2 17.8-8.8
nonextractable 12.5-4.1 11.2-3.4 24.2-7.0
BaP 79.4-7.4 63.3-15.1 46.7-18.4
Based on % total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,3,2 respectively - tic data.
118
-------
c. Influence of SO? and CAP Administration to IPL on BaP
Metabolism
1) Rate of Metabolism
CAP on the IPL acts to slightly inhibit the rate of metabolism of
BaP (Tables 43,46). S02, added concurrently with BaP and CAP to the IPL,
does not change the rate of metabolism when compared to CAP and BaP on the
IPL. This may be due to either the slow leaching of BaP from CAP or an
increase in phagocytic action of the macrophage which might serve to
decrease the amount of BaP available for metabolism. Additionally, at
these low concentrations of SOa, the gas could very possibly be adsorbed
by the particulate and is not available to affect the metabolism of BaP.
The distribution data due to the particulate indicate an increase in
the 7,8- and 9,10-dihydrodiols and a decrease in the nonextractables, and
the monohydroxylated and dione metabolites. It should be noted that the
7,8-dihydrodiol spot contains some (possibly glutathione) conjugation.
This suggests that particulate affects the epoxide hydrase pathway
(37,39,72,23) more readily than the hydroxylation and/or isomerization and
some conjugation pathways. However, when the distribution data for S02,
CAP, and BaP or the IPL are compared to BaP and CAP alone, there do not
seem to be any significant differences except for the 7,8-dihydrodiol,
which contains some unknown metabolite (possibly conjugate), and the
4,5-dihydrodiol. This suggests that S02 at these concentrations does
not cause any marked changes in the metabolic pathways in the presence
of BaP and CAP.
2) Distribution of BaP and Its Metabolites in Tissue at
180 Minutes
There is an increase in metabolism of BaP at 180 minutes when CAP
is administered with BaP compared to the control (Tables 44,45). This
would suggest that after an equilibration time, BaP is leached at a
faster rate. This is consistent with a relative decrease of BaP in the
blood at 180 minutes compared to control, while the lung and trachea
bronchi show a relative increase in metabolite and a decrease in BaP- The
macrophage and washout both show increases in the relative amounts of
the BaP metabolite content. The only significant changes that are
observed in their metabolite profiles are the 7,8- and 9,10-diols and
the nonextractables for the washout fraction.
When CAP and S02 are administered together with BaP on the IPL, there
appears to be an even more dramatic increase in the metabolism of BaP
after 180 minutes compared to CAP and BaP alone (Table 44,45). There also
appears a change in the rate of metabolism of BaP due to CAP plus SO2 as
seen from the results in Tables 43 and 44. Initially the BaP may not be
leached readily from CAP and, therefore, is not available to be metabo-
lized. However, after a period of time, the BaP is probably leached from
119
-------
the CAP at a faster rate. By this time, the S02 has been an effect on
the lung such as to cause cellular injury and/or a change in normal
enzyme functions. These changes result in an increase in the amount of
BaP metabolized.
These observations are reflected in a large increase of metabolite
in blood, trachea bronchi, and lung, a large decrease in metabolite in
washout, a large decrease of BaP in macrophage and washout, and an in-
crease in BaP in the lung compared to CAP and BaP alone. These data are
consistent with relative increases in diol and quinone formation in
blood, macrophage, washout, and trachea bronchi.
120
-------
TABLE 43.
INFLUENCE OF S02 AND CAP ADMINISTERED TO IPL ON BaP METABOLISM
RATE AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animals
Total rate of appearance
of metabolites+in blood
(ng/hr/g lung - S.E. )
Metabolic pattern in
blood (% - S.E.)C
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
Monohydroxylated
Diones
Nonextractables
None
BaP
9
256-38
6.6±0.9d
15.4-4.0
3.3-0.6
9.7±1. I6
10.6-1.8
54.4-5.4
None
BaP+CAPa
5
156-42
19.1-4.4
28.3-7.9
3.0-1.3
5.1-1.4
5.2-2.6
39.3-13.8
None
BaP+CAP+S02b
7
177-48
11.0-1.6
26.7-2.9
1.7-0.7
6.3-1.1
6.9-2.0
42.4-2.6
All metabolites separated by tic. Columns 1 & 3 compared to Column 2.
al mg/kg
bl mg/kg CAP, 2.1 - 0.1 ppm S02
°Metabolite pattern values expressed as percent of total rate of
appearance of metabolite in blood - S.E.
dP - 0.01
eP - 0.05
(Statistics performed by Student-Newman-Keuls Test)
121
-------
TABLE 44.
INFLUENCE OF S02 AND CAP ADMINISTRATION TO IPL ON BaP METABOLISM*
% OF TOTAL BaP AND TOTAL METABOLITE REMAINING IN EACH TISSUE
AT 180 MINUTES - S.E.
Pretreatment
IPL
No. of Animals
% Unmetabolized BaP
% of Total Compound as
Metabol ite in Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound as
BaP in Tissue
Blood
TB
MAC
WO
Lung
BaP
3
65.3 - 4.3
15.5 - 5.2
3.7 - 1.2
0.3 - 0.1
3.3 - 2.3
12.0 - 1.9
12.7 - 1.4
10.4 - 2.7
8.7 - 7.4
2.5 - 1.1
30.9 - 3.6
BaP+CAP
2
40.0 - 26.3
16.6 - 6.7
6.2 - 2.8
1.9 - 1.3
13.2 - 3.3
22.1 - 9.1
1.0 - 0.8
2.2 - 1.0
17.4 - 12.2
10.9 - 9.2
8.5 - 2.5
BaP+CAP+S02
2
18.5 - 8.6
26.7 - 11.4
12.6 - 7.9
2.9 - 0.9
4.7 - 0.4
34.5 - 6.9
1.2 - 0.1
1.1 - 0.1
2.2 - 0.7
0.2 - 0.0
13.8 - 6.3
*tlc data
122
-------
TABLE 45.
% DISTRIBUTION PATTERN OF BaP & METABOLITE IN EACH TISSUE
LUNGX
Pretreatment None None None
IPL BaP BaP+CAP BaP+CAP+SO,
7,8-dihydrodiol 1.2-0.1 6.1-0.5 3.8-0.7
9,10-dihydrodiol 2.9-1.5 3.2-0.2 3.9-0.3
4,5-dihydrodiol 0.9-0.4 1.3-0.2 0.5-0.0
monohydroxylated 10.1-0.6 3.4^0.7 5.6-2.5
diones 1.1-0.3 1.3-1.0 3.4-0.1
nonextractable 5.3-0.9 24.7-2.1 9.6-2.2
BaP 78.5-2.7 60.0-3.2 73.2-5.5
x +
% Total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,2,2 respectively - tic data.
(continued)
123
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4 ,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 45.
(continued)
180 BLOODX
None
BaP
2.8-0.5
6.5-3.8
1.2-0.7
2.5-0.9
1.3-0.1
18.6-2.5
67.0-6.5
None
BaP+CAP
8.0-5.8
5.6-3.4
0.9-0.6
2.2-1.8
1.5-1.3
59.7-33.7
22.1-20.8
None
BaP+CAP+S02
8.0-2.5
16.6-5.2
0.8-0.0
3.6-1.9
5.9-2.7
36.5-1.2
28.6-8.4
x% Total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,2,2 respectively - tic data.
(continued)
124
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 45.
(continued)
MACROPHAGEX
None
BaP
1.4-0.4
0.9-0.2
0.2-0.2
0.9-0.3
0.5-0.2
1.9-1.0
94.2-2.2
None
BaP+CAP
1.5-0.5
0.3-0.2
0.1-0.0
0.2-0.1
0.2-0.1
1.4-0.3
96.3-1.3
None
BaP+CAP+S02
8.1-2.1
0.9-0.0
0.2-0.1
0.5-0.1
4.2-1.9
1.0-0.1
85.0-0.2
X +
% Total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,2,2 respectively - tic data.
(continued)
125
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 45.
(continued)
WASHOUTX
None
BaP
5.0-1.8
10.8-8.3
0.8-0.6
2.7-2.0
2.1-1.3
4.
17.5-6.6
61.0-7.7
None
BaP+CAP
28.4-19.6
5.3-0.5
0.9-0.3
3.2-2.1
2.4-0.6
4.
oToo
59.9-22.1
None
BaP+CAP+S02
20.0-10.1
9.6-6.0
0.9-0.5
3.1-1.8
8.4-6.5
4.
33.4-3.0
24.5-1.6
X +
% Total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,2,2 respectively - tic data.
(continued)
126
-------
Pretreatment
IPL
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
monohydroxylated
diones
nonextractable
BaP
TABLE 45.
(continued)
TRACHEA BRONCHI
None
BaP
0.8-0.2
2.2-1.2
0.9-0.5
3.4-1.8
2.1-1.4
12.5-4.1
79.4-7.4
X
None
BaP+CAP
3.5-1.2
2.6-1.7
0.4-0.3
0.3-0.3
3.5-1.5
26.7-16.1
63.1-21.0
None
BaP+CAP+S02
2.9-0.8
4.0-0.6
0.3-0.1
4.1-2.7
17.8-8.8
24.2-7.0
46.7^18.4
x% Total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,2,2 respectively - tic data.
127
-------
TABLE 46.
INFLUENCE OF S02 + CAP ADMINISTERED TO IPL ON BaP METABOLISM RATE
AND PATTERN OF METABOLISM IN THE BLOOD
Pretreatment
IPL
No. of Animals
Total rate of appearance
of metabol i tes ,in blood
None None
BaP BaP+CAP+S02a
4 7
+ r +
334-40C 191-53
(ng/hr/g lung - S.E.)
Metabolic pattern in
blood (% ± S.E.)b
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4 ,5-quinone
1 ,6-6,1 2-3,6-quinone
4,5-epoxide
9,6-OH
7-OH
3-OH
S Metabolite
Nonextractable
20.1-6.4
2.8-0.4
2.7-0.6d
1.4-0.6
2.3^0.6°
2.1^0.9
2.eio.3g
9.4^3.8
54.3-7.7
17.4-3.5
3.5-0.5
5.7-0.7
4.3-1.9
5.0-0.9
5.9-0.6
2.5-0.4
3.0-0.6h
2.8-0.8
5.9-1.1
43.9-4.0
All metabolites separated by HPLC.
al mg/kg CAP, 2.1 - 0.1 ppm S09.
h
Metabolite pattern values expressed+as percent of total rate of
appearance of metabolites in blood - S.E.
CP = 0.1
dP = 0.05
eP = 0.01 (Student-Newman-Keuls Test)
14- counts appear under this peak.
97-OH and 3-OH collected together
97-OH and 3-OH combined 2.9 - 0.5
128
-------
All of these data suggest that CAP and SO?, administered together
with BaP on the IPL, cause an increase in the rate of metabolism after an
equilibrium period compared to CAP and BaP alone. This is reflected in
more metabolite in blood, trachea bronchi, and lung at 180 minutes.
Summary
The particulate inhibits the rate of appearance of metabolites of
BaP observed in the blood. This can be explained by the relative increase
of BaP and metabolite in the macrophage and washout with corresponding
decrease of BaP in the lung and trachea bronchi. The BaP adsorbed par-
ticulate can be engulfed more readily by macrophages and, therefore, BaP
is less biologically available for metabolism or is leached more slowly
from the lung. However, at the end of 180 minutes the relative amount of
BaP metabolized by CAP plus BaP is greater than the control which is
consistent with an increase of metabolite in lung and trachea bronchi, as
well as washout and macrophage. This then might suggest that CAP which
contains a variety of PAHs and metals might induce the enzyme system
slightly after a period of time for equilibration or that macrophage meta-
bolize BaP but delay the excretion of these metabolites into the blood.
S02 added concurrently with BaP and CAP on the IPL does not change
the rate of metabolism when compared to BaP and CAP alone. This can be
due to slow leaching of BaP or phagocytic action as described previously.
Additionally, at low concentrations of S02 the gas could very possibly
be adsorbed by the particulate and not be available to affect the
metabolism of BaP. After 180 minutes, however, there appears to be a
dramatic increase in the metabolism of BaP. This is reflected in more
metabolite in blood, trachea bronchi, and lung at 180 minutes. This
can be attributed to leaching of BaP from the CAP at a faster rate and
possibly to an effect on the lung by S02 after an equilibration period.
d. HPLC Distribution Pattern for S02 Data
The distribution pattern of BaP and metabolite for S02 data by HPLC
are shown in Tables 47 and 48. There are no major changes except for the
7,8-dihydrodiol on tic which has been broken down into the S metabolite
and the 7,8-dihydrodiol on HPLC (Table 48). There are a few changes in
the total dione and phenol percentage, but that is not completely unex-
pected. On the whole, the HPLC complements the tic data but at the same
time indicates the tic is still a very valid method for analysis of BaP
metabolites.
129
-------
TABLE 47.
EFFECTS OF S02 AND PARTICULATE ON BaP METABOLISM*
% OF TOTAL BaP AND TOTAL METABOLITE REMAINING IN EACH TISSUE - S.E,
Pretreatment
IPL
No. of Animals
% Unmetabollzed BaP
% of Total Compound as
Metabolite in Tissue
Blood
TB
MAC
WO
Lung
% of Total Compound as
BaP in Tissue
Blood
TB
MAC
WO
Lung
so2
BaP
3
44.4 - 4.1
21.6 - 5.1
5.8 - 1.3
0.7 - 0.1
2.3 - 0.3
25.2 - 5.7
10.7 - 3.2
2.4 - 0.7
1.8 - 0.4
2.8 - 0.2
26.6 - 3.0
S02 + BaP
3
49.1 - 0.6
22.4 - 2.1
2.1 - 0.6
1.1 - 0.2
2.8 - 1.1
22.5 - 2.9
15.1 - 6.0
2.9 - 0.5
3.4 - 1.2
3.2 - 0.9
24.5 - 2.2
S02+BaP+CAP
2
18.4 - 10.6
25.6 - 11.1
10.1 - 3.5
7.4 - 3.8
4.1 - 0.6
34.4 - 0.2
0.8 - 0.5
1.3 - 0.7
2.1 - 0.9
0.1 - 0.1
14.1 - 7.5
*HPLC data includes S value.
130
-------
TABLE 48.
DISTRIBUTION PATTERN OF BaP & METABOLITE IN EACH TISSUE
Pretreatment
IPL
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-quinone
4,5-epoxide
9,6-OH
7-OH
3-OH
Metabolite S
BaP
Nonextractable
LUNGX
so2
BaP
1.3-0.5
0.7-0.1
0.7-0.2
0.9-0.1
3.7-0.3
1.0-0.3
0.8-0.2
0.4-0.1
0.3-0.1
2.4-0.4
77.3-2.2
10.4-0.7
-
S02+BaP
5.9-0.8
0.9-0.3
1.6-0.5
0.9-0.4
1.7-0.8
1.3-0.4
1.8-0.1
1.2-0.2
2.3-1.1
3.3-1.2
69.0-1.8
10.0-3.4
-
S02+BaP+CAP
3.6-0.8
1.1-0.4
0.8-0.2
0.8-0.5
1.2-0.4
0.7-0.4
1.0-0.3
1.3-0.7
3.1-1.6
6.3-5.2
70.2-12.0
9.9-2.3
*120 min.
x +
% Total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,3,2 respectively - HPLC data.
(continued)
131
-------
Pretreatment
IPL
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-quinone
4,5-epoxide
9,6-OH
7-OH
3-OH
Metabolite S
BaP
Nonextractable
*120 min.
x% Total activity
Number of animals
TABLE 48.
(continued)
180 BLOODX
so2*
BaP
2.1-0.8
0.9-0.3
1.1-0.1
1.0-0.1
3.2-0.2
1.2-0.2
1.0-0.2
0.5-0.0
0.4-0.2
6.7-4.7
60.0-4.9
23.6-3.2
at 180 minutes in each
are 3,3,2 respectively
(continued)
132
-
S02+BaP
10.1-2.6
1.7-0.5
2.3-0.6
1.6-0.5
2.1-0.7
1.6-0.4
0.9-0.5
0.7-0.1
0.7-0.5
4.8-2.2
53.3-9.8
20.2-9.8
tissue - S.E.
- HPLC data.
-
S02+BaP+CAP
11.3-6.
4.0-1.
4.1+1.
3.2-1.
3.2-0.
2.4-2.
2.9-1.
0.6-0.
3.4-0.
3.0-0.
16.2-3.
45.4-6.
9
3
7
8
8
4
4
6
0
2
3
6
-------
TABLE 48.
(continued)
Pretreatment
IPL
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-quinone
4,5-epoxide
9,6-OH
7-OH
3-OH
Metabolite S
BaP
Nonextractable
MACROPHAGE*
so2
BaP
0.7-0.2
0.5-0.1
0.5-0.0
0.7-0.1
1.2-0.2
0.6-0.3
0.6-0.0
0.7-0.1
0.9-0.4
2.9-0.4
89.4-0.3
1.5-0.2
-
S02+BaP
2.1-0.1
0.9-0.5
0.8^0.3
1.2-0.8
1.0-0.7
1.4-0.5
1.1-0.3
0.8-0.2
1.3-0.5
3.9-1.8
83.7-6.2
1.9-0.7
-
S02+BaP+CAP
1.1-0.2
1.4-1.0
1.0-0.6
0.6-0.3
2.1-1.9
0.9-0.6
1.5-0.7
1.1-0.4
1.0-0.1
7.0-4.6
81.2^4.3
1.1-0.2
*120 min.
x% Total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,3,2 respectively - HPLC data.
(continued)
133
-------
Pretreatment
IPL
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-quinone
4,5-epoxide
9,6-OH
7-OH
3-OH
Metabolite S
BaP
Nonextractable
*120 min.
x% Total activity
Number of animal
TABLE 48.
(continued)
WASHOUTX
so2
BaP
2.3-1.0
0.7-0.1
0.9-0.3
1.1-0.1
1.4-0.5
0.7-0.2
1.1-0.7
1.2-0.4
1.3^0.2
3.2-0.9
78.8-3.7
7.4-2.3
at 180 minutes in each
s are 3,3,2 respectively
(continued)
134
-
S02+BaP
14.2-0.9
1.1-0.3
2.1-0.5
1.2-0.6
0.9-0.6
0.9^0.2
2.0-0.4
1.0-0.3
2.0-0.7
6.4-3.6
60.7-8.3
7.9-2.1
tissue - S.E.
- HPLC data.
-
S02+BaP+CAP
11.3-4.2
3.4-1.5
2.0-0.2
1.7-1.2
5.3-2.0
2.8-2.3
2.0-0.1
1.6-0.7
1.3-0.7
5.4-0.2
17.3-7.6
44.0^11.1
-------
TABLE 48.
(continued)
TRACHEA BRONCHIX
Pretreatment
IPL
9,10-dihydrodiol
4,5-dihydrodiol
7,8-dihydrodiol
4,5-quinone
3,6-quinone
4,5-epoxide
9,6-OH
7-OH
3-OH
Metabolite S
BaP
Nonextractable
so2
BaP
1.9-0.6
0.9-0.3
1.5-0.6
1.3-0.2
2.2-0.5
0.8-0.1
0.7-0.1
0.7-0.1
0.4-0.1
5.1-1.9
55.8-3.5
22.8-6.6
-
S02+BaP
3.4-0.9
0.9-0.1
0.9-0.1
0.5-0.0
0.8-0.2
0.8-0.1
0.6-0.1
0.5-0.2
0.7-0.2
4.9-0.6
74.8-4.4
11.3-3.4
-
S02+BaP+CAP
3.2-0.2
1.7-0.7
2.7-1.5
1.5-0.4
3.0-2.5
2.4-1.0
0.4-0.4
0.9-0.3
3.0-0.5
8.7-5.7
45.6-5.6
26.9-5.4
*120 min.
x% Total activity at 180 minutes in each tissue - S.E.
Number of animals are 3,3,2 respectively - HPLC data.
135
-------
F. DISCUSSION
1. Perfusion - Basic Requirements
Investigations of benzo(a)pyrene metabolism have indicated that the
metabolites are distributed differentially when comparing plasma to
red cells (7). This observation reflects the importance of using whole
blood when possible, since distribution, absorption, and excretion
kinetics are important parameters in estimating total toxicity of a
chemical. The possibility exists where significant factors may be over-
looked when organs are perfused with artificial media. Another reason is
that this perfusate is perhaps the best physiological and biochemical
medium available, i.e., the essential cofactors, trace metals, and autol-
ogous protiens are present. However, the design of the experiment may
dictate the use of artificial media, as for example in the study of lipid
metabolism.
Constant blood pressure and flow are essential for kinetic studies.
The system is essentially chemically and biochemically inert utilizing
silicone rubber tubing and silicone coated glass. The system is operated
at 37°C, and ventilation is accomplished through subatmospheric alterna-
ting pressures. The pH of the blood is controlled through infustion of
NaHCO (0.3 meq/hr) and titrating to pH 7.40 with carbon dioxide added to
the ventilating gas. The materials that are available for analysis
include blood, lung washings, alveolar macrophages, trachea bronchi,
peripheral lung tissue, and ventilating gases.
A summary of the biochemical changes found in the plasma of eight
control isolated perfused rabbit lungs are found in Table 49. One of the
most notable changes is the glucose concentration. The average disappear-
ance rate was approximately 35 mg-%/hr. Infustion of this amount
resulted in no net change throughout the perfusion. Lactate dehydrogenase,
glutamic oxalacetic transaminase (GOT), and lactic acid were found to
increase quite substantially. The increases in LDH and GOT and the
increases in plasma hemoglobin were attributed to hemolysis (44).
Typical physiological values obtained from control lungs are shown
in Table 50. Historically, edema has been a problem in the IPL. Mini-
mization of edema has been evidenced by the small increases in weight of
the lungs measured before and after perfusion.
Cervical dislocation was used to kill the animal since anesthesia
was undesirable for metabolic studies; the location of the strike must
be precise, since brain trauma results in massive hemorrhage and edema
in the lungs. More recently, the lungs have been removed following CO 2
inhalation. We have also noted that in experiments in which the blood
flow decreases with time in the IPL, edema and hemorrhage usually follows.
Corrections were made on the blood flow problems that normally are found
with the IPL hoping that these corrections would influence edema
formation.
136
-------
Figure 11 shows typical blood flow values which we considered
inadequate for metabolic studies before the corrective measures were made.
It was evident that in 1-2 hours a consistent phenomenon was occurring.
Areas of hemorrhage and low perfusion were very evident in these lungs.
Administration of regular maintenance doses of heparin (200-500 IU) as
well as epinephrine (40-100 yg) were found necessary to maintain constant
blood flow in the isolated perfused rabbit lung (Figure 12). Figure 12
illustrates the effects on blood flow of benzo(a)pyrene (BaP) in an
ethanol saline (1:1, v/v) suspension administered intratracheally to the
IPL. The initial decrease in flow is due to the ethanol administration.
Histopathological examination of control lungs revealed no edema and
excellent maintenance of pulmonary structures after 4 hr. of perfusion.
137
-------
TABLE 49.
BIOCHEMICAL CHANGES IN THE PLASMA FROM
BLOOD PERFUSING THE ISOLATED LUNG
Mean Concentrations Change in Mean
in Plasma Concentration
Prior to Perfusion Per Hour
calcium (mg %}
inorganic phosphate (mg %)
glucose (mg %)
adding 30 mg/hr
blood urea nitrogen (mg %)
uric acid (mg %)
cholesterol (mg %)
with Vitamin E
total protein (gm %}
al bumin (gm %}
total bil irubin (mg %}
alkaline phosphatase (mU/ml )
with Vitamin E
lactate dehydrogenase (mU/ml )
SGOT (mU/ml)
plasma hemoglobin (mg %)
lactic acid (mg %)
pyruvic acid (mg %)
13.8*0.8
4.1*0.4
, 236*35
17.5-3.2
0.62*0.18
37-13
5.7-0.4
0.53-0.07
0.14*0.06
55*36
135*42
58-23
0.19*0.12
173-17
1.19-0.06
dec.
inc.
dec.
inc.
inc.
inc.
inc.
inc.
dec.
inc.
inc.
inc.
inc.
inc.
inc.
inc.
N.C.*
0.15*0.29
0.78*0.27
34.5*4.1
±2.3
0.14*0.22
0.20*0.12
4.2*0.7
19.5*7.1
0.10*0.15
0.04*0.03
0.12*0.16
4.8*2.1
0.6*1.1
485*201
121*58
2.3*0.6
19.8*5.2
*0.01
*No change
138
-------
TABLE 50.
PHYSIOLOGICAL VALUES IN THE ISOLATED PERFUSED LUNG PREPARATION
hematocrit (%)
weight gain (%/hr)
blood flow (ml/min)
Pn (mm Hg)
U2
PCQ (mm Hg)
pH range
tidal volume (ml )
mean value = 35.0-5.0; mean change/hr. =
dec. 1.6*0.3
inc. 2.81*1.36
approx. 160-210 (constant in each experiment)
typical values 118*6; 121*10
typical values 39*4; 32*4
7.38 - 7.42
typical values 11.7*0.3; 11.0*0.4
published values 23.9*5.5 (Caldwell & Fry, 1969)
139
-------
FIGURE 11
Inadequate Blood Flow Rate
175 -
Time (hrs)
140
-------
FIGURE 12
Blood
Flow
(ml/min) I4°
1 70
1 60
ISO
140
130
0
H
- 1
1
H HE
1 11
V
1
2
Time
1 •• • — M • ••
H •= H»porln
E c Eplntphrlnt
1 I
3 4
(hrs)
Blood Flow Rate After
Addition of Heparin and Epinephrine
200 -
I 90
B lood i BO
Flow
(ml /min)
170
160
1234
Time (hrs)
Typical Blood Flow Rate
With BaP, Heparin and Epinephrine
Addition
141
-------
2. Control Animals With BaP on IPL
Table 51 shows the distribution of radioactivity in the tissue of
the isolated perfused lung preparation 1 and 3 hours after intratracheal
administration of BaP. After 1 hour of perfusion, approximately 94% of
the total activity remained in the lavaged lungs. At 3 hours of per-
fusion, the proportion of total activity in the lavaged lungs had
decreased to approximately 60%. Plasma and erythrocytes combined con-
tained only 4.5% of the total activity at 1 hour, while at 3 hours these
same two components accounted for approximately 24% of the total activity.
The red blood cells always contained about 50% more activity than the
plasma. Pulmonary alveolar macrophages and the lavage fluid used in
harvesting these cells had only 1% of the total radioactivity at 1 hour
of perfusion. Three hours after addition of the BaP to the preparation,
this distribution had increased to 15% of the total. Based on wet
weight, the pulmonary alveolar macrophages had approximately twice the
activity as the lavaged lung tissue.
The compounds recovered from these fractions included BaP, 3,6-
quinone or dione; and its probable metabolic precursor, 3-hydroxy-BaP.
In addition, three dihydrodihydroxys or dihydrodiols were tentatively
identified on the chromatograms. The K-region 4,5-dihydrodiol was
present. Two others were identified by their similarities to published
R! values and qualitative fluorescent characteristics as the 7,8-dihydro-
diol and the 9,10-dihydrodiol (tic data).
A polar compound or compounds constituted approximately 40% of the
total metabolites in the blood after 3 hours of perfusion. A time-
course study using whole blood revealed that the extraction efficiency of
the solvent was decreasing with time. Exhaustive solvent extraction with
various types of solvent systems did not greatly increase the amount of
radioactivity recoverable from the blood. Therefore, it was assumed that
this radioactivity, which increased logarithmically with time, could be
attributed to a very polar metabolite or metabolites. B-Glucuronidase
did not affect its recovery. Digestion in 1 N HC1 doubled the amount of
extractable radioactivity and sulfatase incubation increased the
extractable activity to comparable levels. However, substantial amounts
of radioactivity remained. The radioactivity remaining after acetone:
benzene extraction was with the polar metabolites. Further identifica-
tion of this metabolite or metabolites is still in progress.
Table 52 shows the distribution of BaP and its metabolites in the
tissue of the isolated perfused rabbit lung preparation 3 hours after
the intratracheal addition of BaP. Control studies using circulating
blood without the lungs yielded only the 1,6-dione, a chemical oxidation
product of BaP, at only about 0.6%/hour. None of the other metabolites
were encountered.
142
-------
In the perfused lung preparation, very little parent compound
(11021% of total) was found in plasma or erythrocytes; most was in the
lungs and lavage fluid. However, whole blood did contain approximately
40% of the total metabolite produced by the lungs after 3 hours. About
45% of this metabolite remained in the lavaged lungs and the remaining
15% was found in the cell-free lavage fluid. This fluid had proportional
amounts of individual metabolites, total metabolites, and BaP, which was
very similar to those seen in lavaged lung tissue. Harvested alveolar
macrophages contained only a small amount of metabolites and a much
larger percentage of unmetabolized BaP.
3. Perturbations With IPL
A summary of the perturbations that have been completed or planned
are presented in Table 53. This table reflects some of the potential
uses of the IPL in characterizing the effects of many environmental con-
taminants of pulmonary metabolic activity. In addition, concurrent
administration of multiple agents are made possible with this system for
the purpose of investigating combined effects of agents in different
physical forms.
143
-------
TABLE 51.
DISTRIBUTION OF METABOLITES IN LUNG
Compound
Total Metabolite3
Polar
7,8-dihydrodiol
9,10-dihydrodiol
4,5-dihydrodiol
3-hydroxy
3,6-dione
B(a)Pa
Total Metabolite, ug
B(a)P, ug
PI asma
89.1
51.2
6.5
25.9
1.7
2.7
1.1
10.9
16.7
2.0
Erythro-
cytes
78.9
46.3
2.4
14.6
4.3
8.0
3.3
21.1
27.1
7.3
Tissue
Alveolar
Macrophage
10.6
3.2
1.2
1.4
0.7
2.2
1.9
89.4
1.2
10.1
Lavage Fluid
(cell -free)
42.1
21.2
2.9
8.4
2.3
7.3
57.9
15.9
21.9
Lung
39.3
18.9
6.2
6.4
0.5
7.3
60.7
48.3
74.7
Values represent percent of total activity in the tissue.
144
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TABLE 52.
DISTRIBUTION OF RADIOACTIVITY
(% of total)
plasma
RBC
lung
alveolar macrophage
lavage fluid
(cell free)
1 hr.
1.86
2.87
94.10
0.89
0.28
3 hr.
9.10
14.90
60.65
5.57
9.78
TOTAL 100.00 100.00
145
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TABLE 53.
PERTURBATIONS PRIOR TO PERFUSION
1. Enzyme Inducing Agents (ip)
PB, 3-MC, B(a)P, PCB's
2. Inhalation Exposure
SOp, n-dodecane, coal dust, metals
3. Dietary Manipulations
4. Intratracheal Instillations
B(a)P, crystalline quartz, papain, asbestos
Ferric Oxide
Crude Air Particulate (CAP)
CONCURRENT ADMINISTRATION OF MULTIPLE AGENTS TO IPL
CAP + B(a)P - S02
Ferric Oxide + B(a)P
Ethanol + Trichloroethylene
146
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4. Effects of Enzyme Inducers
Phenobarbital does not induce the aryl hydrocarbon hydroxylase
enzyme system (P^SO). Corn oil, on the other hand, is significantly
different from the control which indicates that corn oil does induce the
enzyme system. The metabolic profile for corn oil and the control are
similar with less phenol and quinone and more 7,8-dihydrodiol formation
for corn oil pretreatment.
Both BaPjp and BaPjj pretreatment cause increases in the rate of
appearance of BaP metabolites in the blood. This is due to the fact that
BaP will induce the P^BO enzyme system (64). The metabolic profile shows
a marked increase in the 9,10-dihydrodiol for BaPjp or BaPjj pretreatment
while there is a significant decrease in the mononydroxylated and dione
for BaPjj from control and BaPjp pretreatment.
There are also large changes in the distribution of BaP and its
metabolites in the blood and lung. With concomitant increases in total
metabolite in the blood and lung, there are corresponding decreases of
BaP in these tissues. These major changes are consistent with increases
in nonextractables in the blood and lung and with increases in the
9,10-dihydrodiol in the blood. This suggests that as more intermediate
epoxides are formed at a faster rate, they are converted into conjugates
of BaP or bound to macromolecules (64). Additionally, as the pathway
becomes saturated the epoxide hydrase converts the 9,10-epoxide to
9,10-dihydrodiol and excretes it into the blood in both cases and
rearrangements and/or isomerization converts some intermediates to
phenols and quinones in BaPjp pretreatment.
These data suggest that even though BaP is given by two different
rates of administration, the rates of metabolism are significantly higher
than the control in both cases and similar enzyme systems are induced.
In one case, the enzyme levels are increased in whole animal but
specifically in the liver 24 hours later, while in the second case the
enzyme levels are increased in the lung specifically over a six week
period.
SMC is not as good an enzyme inducer (Pi450) as BaP as indicated by
the total rate of metabolism. The total rate can partially be accounted
for by the corn oil administration. SMC definitely causes an increase
in the rate of metabolism compared to the corn oil and control, but is
not significantly different at the 90 percent significance level (60).
The metabolic pattern indicates that SMC and BaP have similar profiles
which are different from the corn oil control. SMC and BaP appear to
stimulate 9,10-dihydrodiol production.
147
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These data for SMC and BaP suggest that the metabolite patterns
produced by PI 450 enzyme inducers are similar and that more polar material
is excreted into the blood stream than the appropriate controls. The
turnover rate is faster and, therefore, the increased amount of epoxides
that are formed as intermediates rearrange or isomerize to phenols and
quinones or open up to 9,10-dihydrodiols by epoxide hydrase action.
This system, therefore, appears to be a good model system for
stydying the metabolism of BaP. In recent studies (49,62) incubation of
BaP in human and rat lung microsomes produced 30% and 20% total dihydro-
diol respectively. This is consistent with our results for rabbit IPL
in the production of 25-30% dihydrodiol formation depending on
experimental conditions (68).
5. Particulate Effects
When particulate is administered with BaP on the IPL, the particulate
inhibits the rate of appearance of metabolites of BaP observed in the
blood. This can be explained by the relative increase of BaP and
metabolite in the macrophage and washout with corresponding decrease of
BaP in the lung and trachea bronchi. The BaP adsorbed particulate can
be engulfed more readily by macrophages and, therefore, BaP is less
biologically available for metabolism or is leached more slowly from
the lung. However, at the end of 180 minutes the relative amount of BaP
metabolized by CAP plus BaP is greater than the control which is con-
sistent with an increase of metabolite in lung and trachea bronchi, as
well as, washout and macrophage. This, then, might suggest that the
CAP which contains a variety of PAHs and metals might induce the enzyme
system slightly after a period of time for equilibration or that the
macrophage metabolize BaP but delay the excretion of these metabolites in
the blood.
When BaP is given IP to the whole animal, the rate of appearance of
metabolites is increased significantly compared to its control. However,
when CAP is introduced with BaP on the perfusion the rate of appearance
of metabolites is decreased. The rate of appearance and the metabolite
profile are similar to the corn oil pretreatment which indicates that CAP
negates the effect of pretreatment. However, as the perfusion proceeds
through 180 minutes, more BaP is metabolized, i.e. more BaP is. available
by leaching from CAP. In fact, the profiles for BaPip pretreatment
followed by BaP alone and BaP plus CAP on the IPL are similar which
suggests that after equilibration for a period of time, more of the BaP
adsorbed on CAP is available for metabolism. This is reflected in larger
amounts of nonextractables and 9,10-diol excreted into the blood with
corresponding decrease of BaP. However, there are still minor differences
at 180 minutes in that slightly more BaP and less metabolites are found
in macrophage, lung, and trachea bronchi after introduction of CAP. With
additional time, more BaP may become available for metabolism and,
148
-------
therefore, these differences could disappear.
CAP given as a pretreatment acts as an enzyme inducer to increase
the rate of metabolism of BaP. BaP adsorbed on CAP administered as a
pretreatment also acts like an enzyme inducer by increasing the rate of
metabolism but the rate of metabolism is not the sum of the individual
rates. This indicates that BaP is not leached readily from CAP, i.e. BaP
is not available for enzyme induction. Overall CAP or BaP by itself
increase the diol formation in the tissues with smaller increases or
decreases in nonextractables. Together BaP and CAP decrease the diol
formation and increase the nonextractables in the tissues.
When CAP is given as a pretreatment followed by BaP plus CAP on
perfusion, the metabolism of BaP is increased compared to CAP pretreat-
ment alone. Overall, these observations are reflected in decreases
in diol formation and increases in BaP content. The major increase of
BaP is in the macrophage which is consistent with decrease in metabolism;
the BaP adsorbed or CAP administered to the IPL causes an increase in the
rate of action of macrophage (5,19).
CAP appears to act as a cocarcinogenic agent with BaP. CAP acts as
a physical agent in decreasing the biological availability of BaP or in
the slower release of BaP over time when administered on the lung with BaP
in comparison with pretreatment of particulate only. This data suggests
that particulate affects the BaP metabolism by two different mechanisms:
one mechanism appears to be a long-term effect due to pretreatment of
particulate which causes an increase in the total metabolic activity and
the other mechanism is a short-term effect due to particulate being
administered to the IPL which causes a decrease in the total metabolic
activity and inhibits the effects of pretreatment.
This work helps to partially clarify the ideas of a number of
investigators (12,21,53,57,61). Particulates used in maintaining the
environment of BaP in the lung for long periods appear responsible for
increasing tumorigenic responses due to slow release of BaP from particu-
late. Also, particulates appear to influence metabolic pathways. The
results presented indicate that both factors may be responsible, i.e.
slower release of particulate as measured by appearance of metabolites in
blood by IPL and a significant change in metabolic pathways.
With concomitant administration of particulate and BaP and, there-
fore, a slower release of rate, BaP is effectively administered to the
lung tissue in small doses when compared to BaP itself. This type of
treatment with particulate appears to be similar to previous observations:
a carcinogen is much more effective in producing tumorigenic response when
given in small divided doses over a period of time as opposed to single
large equivalent doses.
149
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6. S02 Effects
S02 pretreatment increases the rate of metabolism compared to
control but does not significantly change the metabolite pattern. The
SO pretreatment data, however, are very different from that obtained
for an enzyme inducer which affects not only the rate but also the pat-
tern. This difference in rate between an enzyme inducer, BaP, and S02
is reflected in a large increase in the 9,10-diol a slight decrease in
the nonextractable.
After 180 minutes, similar results are obtained in that the amount
of BaP unmetabolized in each case is consistent with rate of metabolism.
In general, there are some small increases in the amount of nonextract-
ables for S02 pretreatments compared to control while there are some
rather large increases in the nonextractables for BaPjj pretreatments
compared to S02 and control. These data again suggest that $62 pretreat-
ment show an increase in the rate of metabolism but does not significantly
affect the pattern. SOa, therefore, does not appear to affect BaP
metabolism by enzymatic induction.
SOz administered on the IPL with BaP causes an increase in the rate
of metabolism of BaP compared to control but does not significantly
change the metabolic pattern. When CAP and SOz are administered concur-
rently with BaP on the IPL, the rate of metabolism of BaP is significantly
decreased compared to S02 alone and does not change when compared to CAP
alone. This could be due to the fact that BaP is not leached readily from
CAP or to increased phagocytic agent. Additionally, at low concentrations
of S02 the gas could very possibly be adsorbed by the particulate and is
not available to affect the metabolism of BaP. The CAP and S02 together
do cause small changes in the metabolic pattern compared to the control
with increases in the 7,8- and 9,10-diol and a decrease in nonextract-
ables.
At 180 minutes, the amount of unmetabolized BaP left for BaP plus
S02 is consistent with rate of metabolism. S02, in vitro, does not
appear to have major effects on the metabolite pattern. CAP plus S02
administered together with BaP, on the other hand, causes a significant
increase in the rate of metabolism of BaP after an equilibration period.
This can be due to the combination of leaching of BaP from CAP at a
faster rate and changes in normal enzyme functions and/or cytopathological
effects due to S02. Under these conditions S02 may cause an increase in
enzyme activities.
Our attempt is to simulate environmental conditions. The 312 ug of
BaP, 1 mg/kg of CAP, and 1-2 ppm of S02 used in the experiments are
realistic human exposure values; however, there are some limitations in
our system. The short time period will show only an immediate effect;
with S02j CAP, and BaP together, it may be necessary to run the experiment
150
-------
for longer periods of time, perhaps at higher concentrations of S02, or
to use larger amounts of BaP and CAP to obtain dose-response relation-
ships, or to pretreat with S02 by inhalation (9). In the presence of
CAP, the S02 can be adsorbed on particulates and some of the SCL may
be catalytically converted to bisulfates under the right conditions of
temperature and humidity: in the presence-of sunlight, the S02 can be
converted photochemically. We are more interested at this point, however,
in the effects of SC"2 on BaP metabolism under our stricter environmental
conditions (3,10,22,34,38,63).
S02 increases the metabolism of BaP by the I PL, but does not affect
the metabolic pattern. It acts as a biochemical agent which causes
biochemical changes in the lung due to irritation (10,42). More work in
this area needs to be done in order to answer this question. The data
indicate, however, that S02 can produce changes in the rate of metabolism
of a well-defined carcinogen.
The presence of S02 at 1-2 ppm with CAP and BaP together indicates
that 90z is either adsorbed by the particulate or that BaP is not
available for metabolism due to either that BaP is not leached readily
from the particulate, or an increase in phagocytic action of the macro-
phage which might serve to decrease the amount of BaP available for
metabolism. The particulates, it should be mentioned, can maintain the
environment of BaP in the lung for long periods of time which may be
responsible for increased tumorigenic responses and altered metabolic
pathways (12,20,53).
These studies are a first attempt to determine what type of
interactions take place when S02 and/or CAP are added with BaP to a
lung model system. This system appears to be a good model system for
studying the metabolism of BaP in the presence of S02.
151
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disulfitomercurate (11) and subsequent colorimetric estimation.
Analy. Chem.. 28(12):1816-1819.
70 Wislocki, P. G., A. W. Wood, R. L. Chang, W. Levin, H. Yagi,
0. Hernandez, D. M. Jerina, A. H. Conney (1976). High mutagenicity and
toxicity of a diol epoxide derived from benzo(a)pyrene. Biochem.
Biophys. Res. Cornm.. 68(31):1006-1012.
71 Wislocki, P. G., R. L. Chang, A. W. Wood, W. Levin, H. Yagi,
0. Hernandez, H. Mah, P. M. Dansette, D. M. Jerina, and A. H. Conney
(1977). High carcinogenicity of 2-hydroxybenzo(a)pyrene on mouse skin.
Cancer Res., 37:2608-11.
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72 Wood, A. W., R. L. Goode, R. L. Chang, W. Levin, A. H. Conney, H. Yagi,
P. M. Dansette, and D. M. Jerina (1975). Mutagenic and cytotoxic
activity of benzo(a)pyrene 4,5,7,8- and 9,10-oxides and six correspond-
ing phenols. Proc. Natl. Acad. Sci., 72(8):3176-3180.
73 Yang, S. K., H. V. Gelboin, B. F. Trump, H. Autrup, and C. C. Harris
(1977). Metabolic activation of benzo(a)pyrene and binding to DNA
in cultured human bronchus. Cancer Res., 37:1210-1215.
74 Yang, S. K., P. P. Roller, P. P. Fu, R. G. Harvey, and H. V. Gelboin
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75 Young, S. L. (1976). An isolated perfused rat lung preparation.
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158
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-80-029
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Study of the Effect of Whole Animal
Exposure to Acid Mists & Particulates on the Pulmonary
Metabolism of Benzo(a)pyrene in the Isolated Perfused
Lung Model.
5. REPORT DATE
July 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Warshawsky, D, Niemeier, R.W., and Bingham, U.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
University of Cincinnati College of Medicine
Department of Environmental Health
3223 Eden Avenue
Cincinnati, Ohio 45267
10. PROGRAM ELEMENT NO.
1AA817
11. CONTRACT/GRANT NO.
Contract No 68-02-1678
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP,NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
Project Officer: Stephen Nesnow
16. ABSTRACT
Lung cancer represents the highest single
Epidemiological and experimental evidence
environmental factors is responsible for
exposed to a complex mixture of potential
carcinogens and a variety of agents which
disposes of inhaled materials. One such
ubiquitous environmental pollutant formed
coal and in other processes that involve
cause of cancer deaths in the U.S.
indicates that the interplay of multiple
the induction of lung cancer. Man is
ly hazardous materials, including specific
may modify the manner in which the lung
carcinogen is benzo(a)pyrene (BaP) a
during the destructive distillation of
incomplete combustion of organic material.
BaP in combustion with various agents, such as ferric oxide, has been used in
animals to experimentally induce tumors of bronchogenic origin. Evidence describes
the necessity for this compound, BaP, to be metabolized to produce the carcinogenic
response. However, the metabolism of BaP in the lung has not been fully investigated
Since at least three enzymes are involved in the metabolism of this compound and
some of these systems can be inhibited by the presence of Fe203, S02, or CAP to
produce different metabolic patterns, a study of all the metabolites in the lung is
necessary in order to determine if the rate or pattern of formation has changed.
Therefore, an isolated perfused rabbit lung preparation su.itattle for.metabolic
s has hppn Hpvplnnpd tn ctnHw Rap in crude air particuTate and/or 5U2-
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Lung cancer
Benzo(a)pyrene (BaP)
Environmental pollutants
Metabolic patterns
06 F,T
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
159
20. SECURITY CLASS iThis page/
UNCLASSIFIED
22. PRICE
PPA farm 2550-1 (R«v. 4-77) ^PREVIOUS EDITION IS OBSOLETE
159
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