United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-84-023 Jan 1985
&EPA Project Summary
Chromosome Studies on Human
Subjects Exposed to Arsenic in
Drinking Water
Baldev K. Vig
A 2-year study was carried out on
human subjects of various ages and
backgrounds who have been drinking
water containing 0.05 mg/l (0.05
ppm) or more arsenic for a period of at
least 5 years. A control group that had
been exposed to arsenic in drinking
water at concentrations less than 0.05
mg/l was used for some aberrations
and sister chromatid exchanges in the
lymphocytes of the two populations
with the amount of arsenic in the water
they have been drinking. A questionnaire
was designed to explore the incidence
of skin cancer, fetal wastage, any
genetic development abnormalities in
the family and related parameters.
Several other variables, e.g., coffee,
wine and cigarette consumption, sex,
residence (rural vs. urban) and exposure
to chemicals, smelter and pesticides
were taken into consideration. The
study involved the city of and areas in
the vicinity of Reno, Nevada.
This study on chromosome aberrations
(104 exposed and 86 control individuals)
and on sister chromatid exchanges (98
exposed and 83 control individuals) did
not show that arsenic at a mean level of
0.109 mg/l has any effect on these
parameters. Similarly, no effect of
arsenic at these modest concentrations
was found on other health parameters
studied.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory. Research Triangle Park. NC,
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction and Summary
Inorganic arsenicals have been impli-
cated in several health problems A
partial list includes skin-cancers, liver
cirrhosis, gastrointestinal catarrh, hyper-
keratosis, polyneuntis, and genetic
damage. Many parts of the United
States contain rather high quantities of
inorganic arsenicals in their drinking
water, especially obtained from under-
ground deposits Considering the results
from several different studies mostly of
non-U S origin, the United States
Environmental Protection Agency (EPA)
set "Interim Drinking Water Regulations"
in 1976which requirethat drinking water
contain less than 0.05 mg/l of total
arsenic
In the meanwhile, the EPA has initiated
an extensive research program to assess
health effects in relation to various levels
of arsenic in drinking water. Recent in
vitro experiments and study of lymphocytes
of patients treated for psoriasis with
arsenic containing compounds have
shown that arsenic induces genetic
damage Furthermore, a strong correla-
tion exists between carcinogenic and
mutagenic potentials, especially induction
of Sister Chromatid Exchanges (SCEs), of
chemicals With this in view, a study was
initiated m 1978 to find out if any
increase in the frequency of chromosome
aberrations and SCEs is evident in a
population drinking water containing
0 05 mg/l or higher arsenic in comparison
to a population drinking water containing
less than 0 05 mg/l arsenic
The study centered around the city of
Reno and encompassed some neighboring
communities, viz. Hidden Valley, Fallen,
Virginia Foot Hills, and Fernley Most of
the residents of Reno had been drinking
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water containing an average of 0 01 +
0 001 mg/l of arsenic (range <0.005 to
0.045 mg/l) and thus served as control
group. Those of other communities,
especially Fallen — 50 miles NE of Reno
— have an average of 0 109 + 0.005
mg/l of arsenic (range 0.05 to 0 39
mg/l) in drinking water One individual
had been drinking water with 2 275
mg/l arsenic but was not included in
further statistical analysis This group
served as the "exposed" population or
"cases." In order to be included in the
study as control the subject was to have
been drinking water containing <0 05
mg/l arsenic for at least 5 years, and
similarly >0.05 mg/l arsenic for 5 years
to be included as "exposed."
A questionnaire was designed to obtain
data on the following: sex, ethnic origin,
age, occupation, exposure to pesticides
and other chemicals, water quality and
quantity food and drinking habits,
smoking, health status including pains,
liver and kidney disease, nerve disorders,
arthritis, cancer or ulcers, anemia, bone
problems, hair, nail or weight loss,
hyperpigmentation of skin, nausea,
menstrual irregularities, stillbirths,
miscarriages and pregnancy complica-
tions A telephone call was used for the
first contact and participation was
entirely voluntary The questionnaire was
completed by personal interview. At that
time a water sample from the kitchen tap
was obtained to analyze arsenic content
A 5-ml blood sample was obtained for
chromosome studies.
The arsenic content of water was
analyzed by the Nevada State Health
Laboratory using atomic absorption as
well as by colometric technique Chromo-
some preparations were carried out by
culturing whole blood in medium 1A
(GIBCO)for48 to 72 hours ForSCEsthe
cells were treated with BrdU for two
rounds of replication. Routine colcemid,
hypotonic treatments were used for
harvesting Giemsa stain was used on
slides used for chromosome aberrations
and f luoresent plus giesma technique for
SCE analysis. Attempts were made to
analyze up to 200 cells for various types
of aberrations (gaps, breaks, exchanges
or chromatid and chromosome type) and
at least 25 cells for SCE analysis). Quality
control was always exercised to assure
reproducibility of results of all studies.
A total of 211 cases were included in
the study. The population samples in the
control and exposed groups expressed
considerable, though not perfect, simi-
larities in life style Relevant information
for the control (86 volunteers) and
"exposed" (105 volunteers) populations,
respectively, are as follows age in years =
29 56 + 15 vs. 42 20 + 1 6; water
consumed (glasses per day) = 4 35 +02
vs. 5.1 2 + 1.6; coffee consumed (cups per
day) = 3.30 + 0.4 vs. 4.09 ± 0.3; % males =
58 1 vs. 58.3, % exposed to pesticides =
7 0 vs 9 5; % exposed to smelter - 1.2 vs
2 9; % exposed to chemicals = 1 2 vs 29;
% cigarette smokers = 16.3 vs 15 2, %
wine drinkers = 25.6 vs 12.4; % residents
of rural Nevada =31 4 vs. 92.4. Statistical
analysis of the control vs. exposed
population showed no significant differ-
ences with respect to sex, exposure to
pesticides, smelter, chemicals or smok-
ing However, there were more wine
drinkers among control than exposed
individuals (X2 =4.66 on Idf, p =0.03) and
more exposed individuals came from
rural than urban sample(x2= 74 56 on Idf,
p<0.001) The mean age of the control
group (29.6 years) was also significantly
(p<0.001) less than that of the exposed
group (42 2 years). The mean water
consumption or coffee consumption was
not significantly different.
For various reasons, only 191 cases
were found suitable for the two types of
cytogenetic analysis. Of these, 190
cultures used for analysis of chromosome
aberrations were derived from 86 control
and 104 exposed volunteers Two hundred
cells per individual were analyzed in
every case except for 16 cases. The study
of SCEs was based on a sample size of
181 individuals, 83 in control group and
98 in the exposed. A sample of 50 cells
per case was analysed for SCEs for the
first 62 cases and 25 cells per case
thereafter.
Results
A total of 18,240 cells were studied for
chromosome aberrations among the 86
control individuals and 19,264 cells from
the lymphocytes of 104 exposed. The
frequency of chromosome aberrations in
the entire population studied ranged
between 0.00 to 0.13 aberrations/cell,
with a mean of 0.033+0.002 aberrations/
cell in the control population and 0.026 +
0.002 aberrations/cell in the exposed
group. The frequency of aberrations,
though lower in the exposed population
than the control, is none-the-less within
normal range for both The frequency of
SCEs, however, was similar in the two
groups. The range for the entire group
was 5.84 to 15.28 SCEs/cell with an
average of 8.301 + 0113/cell for the
control group and 8.306 + 0.141 /cell in
the exposed group.
Detailed information on multiple
regression analysis of chromosome
aberrations and SCEs as function of
arsenic exposure and other independent
variables is provided in Table 1.
Statistical interpretation of the data
showed that the six multiple regressions
using chromosome aberrations as a
dependent variable were all significant,
with 109%-13.0% of the variance being
explained by the 10 independent vari-
ables as a group. The urban population
sample showed significantly higher aber-
ration frequency than the rural group
There was also some indication that cof-
fee consumption may increase the fre-
quency of chromosome aberrations.
However, arsenic exposure showed no
relationship with chromosome aberra-
tions in our sample.
Using SCE as a dependent variable, all
the six multiple regressions were statis-
tically significant However, only 11.1-
13.6 percent of the total variance in SCE
was explained by the 10 independent
variables as a group Specifically, smokers
exhibited more SCEs than non-smokers
However, arsenic concentration in drink-
ing water showed no effect on the fre-
quency of SCEs
The average arsenic content in the
exposed group in this study is more than
twice (exactly 218%) of the highest limit
mandated by the EPA. The quantity of
water consumed is also higher for the
exposed populations since most of it was
derived from farming communities in
rural Nevada The mean age of the
exposed group is also higher than that of
the control (p<0.001), which may dispose
the exposed group to higher frequency of
chromosome aberration In spite of these
factors, our data do not show an in-
creased incidence of chromosome aber-
rations or SCEs in the population exposed
to modest quantities of arsenic in
drinking water
Conclusions
These studies show that arsenic when
consumed in modest quantities (at least
up to 0.1 mg/l) in drinking water for up to
5 years does not appear to cause any
detectable increase in the frequency of
chromosome aberrations or SCEs in the
lymphocyte cultures Besides, m the
limited population surveyed no evidence
of increased incidence of other serious
health effects were observed.
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Table 1. Summary of Multiple Linear Regression Analyses of Chromosome Aberrations and Sister Chromatid Exchanges as Functions of Arsenic Exposure
and Other Independent Variables
Dependent
Variable
SCE
ARSENIC
061
057
Standardized Regression Coefficients for Independent Variables'
ARSGRP
052
054
ARSWAT
163+
164+
SEX
-097
-098
- 102
-093
-094
-097
AGE
047
049
052
044
044
046
PEST
- 119
- 114
- 136
- 117
- 112
- 134
SMELT
002
007
010
006
011
014
CHEM
-043
-046
-046
-050
-052
-052
COFFEE
151
149
157
LOGCOFF WINEGRP CIGSGRP
030 168+
031 167+
011 170+
129 025 183+
129 026 181 +
139 006 185+
C/TYGRP
134
134
171
129
133
169
F> r'
{For overall
regression!
223+ HO
2 21+ 111
2 65' 13 1
2 12+ 110
211+ 110
255* 130
CRABNOR -013 -003 026 035 057 -066 162+ -058 065 316" 262* 12 8
-007 -003 025 033 056 -066 163+ -058 065 319* 262* 128
040 - 004 023 027 057 - 065 164+ - 062 067 339* 2 65* 13 0
-018 009 067 030 070 -068 028 -042 110 311* 218+ 109
-006 009 065 028 069 -067 028 -042 111 317* 217+ 109
033 008 063 023 070 -066 029 -045 113 334* 219+ 110
ORABLOG -014
-043
-017
-043
020
016
-020
-021
-020
-013
-014
-013
-010
-O07
-013
014
017
010
059
057
054
056
054
052
096
094
095
103
102
103
-071
-072
-070
-072
-072
-071
092
092
093
014
013
015
-062
-063
-063
-052
-054
-053
041
040
043
068
067
069
281*
263*
296*
278
262
293
1 94+
1 96+
1 95+
1 81
1 83
1 81
98
99
98
92
93
92
'Significance levels indicated as follows + -0 01
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Baldev K. Vig is with the University of Nevada, Reno, NV 89557.
Daniel G. Greathouse is the EPA Project Officer (see below).
The complete report, entitled "Chromosome Studies on Human Subjects Exposed
to Arsenic in Drinking Water, "(Order No. PB 85-125 821; Cost: $10.00, subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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