United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-83-003 June 1983
4>EPA Project Summary
Chlorinated Hydrocarbons:
Insecticide Versus Carcinogenic
Action
S. M. D'Ambrosio, N. J. Lewis, R. W. Hart, and W. J. Collins
The purpose of this grant was to
determine those structural character-
istics of chlorinated pesticides
responsible for deleterious as opposed
to beneficial effects. These studies have
led to the development of a model for
the design, synthesis and evaluation of
insecticides with reduced genetic
hazard.
Various halogenated hydrocarbons
and their analogs were designed and
synthesized for this study. The test
systems employed for evaluation of the
effects on mammals and insects were:
a) normal and SV-40 transformed
human fibroblasts; b) DNA repair by
Unscheduled DNA Synthesis (UDS)
and 5-bromo-2'-deoxyuridine (BUdR)
photolysis; c) measurement of associa-
tion of 14C-labeled synthetic analogs
with genetic material; d) cytotoxicity; e)
metabolic activation studies using liver
homogenates; and f) topical and oral
toxicities of standards and test com-
pounds to house flies (Musca domes-
tica) and mosquito larvae (Aedes
aegypti).
These studies indicated that
modification of the 6,7-double bond of
aldrin (and the 6,7-epoxide of dieldrin)
could result in potent insecticides with
reduced mammalian cytotoxicity, DNA
repair, and DNA association. These
studies included the synthesis and
evaluation of three distinct series of
pesticide analogs, including two
series of halogenated cyclodienes with
modified ?r-electron character and a
series of aromatic pyrethrin-related
agents.
This report was submitted in
fulfillment of Grant No. R-805008
under the sponsorship of the U.S.
Environmental Protection Agency
covering the period July 18, 1977 to
July 17,1980. This project represented
an interdisciplinary research study
combining the scientific expertise of
medicinal chemists, cell-molecular
biologists, genetic toxicologists and
entomologists.
This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory. 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
Population growth and an increase in
groups that do not directly participate in
food production have increased the
demand for more efficient agricultural
productivity. This demand has been
satisfied by both higher crop yields and
the elimination of man's natural
competitors through pesticides. Although
some pesticides do not show an immedi-
ate effect in vivo at concentrations
normally used in agriculture, they may
pose a significant long-term hazard to
man. The ideal pesticide should
selectively affect a desired species for a
specified period of time and then
disappear without any trace. However, no
such pesticide currently exists, and the
present policy of pesticide use is a com-
promise between desirable and undesir-
able effects. The purpose of this study
was to develop an approach to evaluating
the relationships between agricultural
insecticides and the induction of genetic
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damage in mammals by identifying those
structural features of the halogenated
hydrocarbon class of pesticides responsi-
ble for the species-specific observed
effects. More specifically, this study:
1) Synthesized a rationally designed,
selected series of compounds with
systematically juxtaposed
functional groups,
2) Evaluated the toxicities of these
chemical agents in a variety of
insect model systems,
3) Evaluated their effects on in vitro
mammalian cells in culture with
respect to cytotoxicity, DNA repair
and DNA replication,
4) Synthesized, 14C-radiolabeled
intermediates of primary interest
to determine the extent and type of
macromolecular interaction. These
radiolabeled compounds were
used to evaluate cellular, genetic,
and metabolic parameters associ-
ated with the pesticide molecules,
and
5) Studied the molecular features of
pesticides that seemed to
contribute to mammalian cell
genetic damage and assessed the
relationship of this damage to
insect toxicity.
Results and Discussion
Synthesis and Selection of
Halogenated Pesticides
The halogenated pesticides evaluated
in this study included numerous EPA
standards and a variety of analogs of the
halogenated polycyclic insecticides,
aldrin and dieldrin. Individual analogs
were synthesized and evaluated for their
insecticidal and mammalian effects. The
metabolically reactive portions of the
aldrin or dieldrin molecules were
chemically manipulated, and several
series of related halogenated cyclodienes
were studied. Additionally, a second
study assessed the effects of chemical
manipulation on extended aromatic and
7r-electron rich moieties in other
categories of pesticides based upon
findings from the aldrin-related series of
molecules. These studies included
numerous analogs and agents related to
aldrin, dieldrin, and chlordane. The
synthetic approaches were extended to
the pyrethrin insecticides based upon
results obtained early in the grant period.
The approaches resulted in the synthesis
of a variety of novel chemical agents with
observed insecticidal properties. Several
of these agents were radiolabeled for
further macromolecular interaction
studies. This led to the synthesis of
chemical agents which retained their
insecticidal action while demonstrating a
markedly reduced deleterious effect on
mammalian systems.
Insect Toxicity Studies
All chemical agents and EPA
standards were evaluated for their
insecticidal action in house fly (Musca
domestics) and mosquito (Aedes aegypti)
test systems utilizing both topical and oral
administration routes. Additionally,
experiments were performed to deter-
mine the synergistic effects of
insecticides and the effects of
metabolism on the observed toxicities of
pesticides from the classes involved in
these studies.
The studies indicated little direct
relationship between high halogen
content, aromatic ring content, and
insecticidal action. The halogenated
cyclodiene analogs demonstrated only
very weak activity when administered
either orally or topically. The compounds
with extended ir-electron rich systems
(utilizing pyrethrin-related agents) failed
to demonstrate decreased mammalian
toxicity while retaining pesticidal action.
Numerous analogs of aldrin and dieldrin
provided effective insecticidal agents and
demonstrated the greatest activities
when used in conjunction with
synergistic agents such as piperonyl
butoxide. The results of these studies
clearly demonstrated that structure-
action correlations could be established
based upon probable metabolic routes in
the insect species selected.
Our studies further demonstrated a
stereochemical selectivity of cis isomeric
pyrethrin-related analogs as the most
effective insecticidal agents. Naturally
occurring and other synthetic analogs
usually favor the trans isomeric struc-
tures. Additionally, the most effective
insecticidal analog of aldrin, 6,7-dihydro-
aldrin was shown to be converted by
mammalian liver homogenates to aldrin,
dieldrin, and 6-hydroxydihydroaldrin.
In Vitro Mammalian
Cellular Effects
The cytotoxic effects of the chlorinated
hydrocarbons synthesized were
determined in normal fibroblasts (C-1 53)
and transformed human fibroblast (VA-4)
cell lines. Aldrin, dihydroxyaldrin and
dieldrin at a 100 uM concentration
reduced the colony-forming ability to 0%
in both C-153 and VA-4 fibroblasts. Othei
structural modifications of the active
moieties of the chlorinated hydrocarbons
greatly reduced cytotoxicity, but not to the
level observed with the above three
compounds. Allethrm and pyrethrin, ol
the pyrethroid class exhibited a high level
of cytotoxicity resulting in 0% survival at a
100 juM concentration in the C-153 and
VA-4 cell lines. All compounds tested
exhibited typical cytotoxicity curves
dependent upon dose.
Induction of DNA Repair
Dieldrin induced UDS in the VA-4 cell
line, whereas it showed comparatively
less UDS in the C-153 and Cl (normal
human skin fibroblast) cell lines. Aldrin
did not show any UDS in the cell lines
tested. Of the structural analogs tested
for UDS, those containing a reactive
moiety at the 6,7 position induced UDS.
The other analogs did not appear to
induce significant levels of UDS. Repair
was also measured using the BUdR
photolysis technique. Aldrin and dieldrin
both showed photolyzable sites m the Cl
cell line, whereas dihydroaldrin did not.
Aldrin and dieldrin both inhibited normal
DNA replication. Pyrethrin stimulated
repair while allethrin and permethrin did
not.
Radiolabeled Agents for
Assessment of DNA-Association
'4C-Dihydroaldrin and 14C-dihydrox-
ydihydroaldrin were synthesized from
14C-aldrin and 14C-dieldrin by novel
synthetic procedures in order to assess
the relationship of pesticidal association
with macromolecular cell components.
Coincident with UDS data, 14C-dieldrin
was found to be associated with VA-4 and
153 cells to the greatest extent. 14C-
Aldrin and 14C-dihydroaldrin did not
associate with the DNA of the VA-4 and
153 cells, whereas, 14C-dihydroxydi-
hydroaldrin did associate to a limited
extent. Activation studies utilizing S-9 rat
liver microsomes increased the binding of
14C-aldrin and 14C-dihydroaldrin in both
VA-4 and 153 cell lines. It was also
demonstrated that 14C-dieldrin associ-
ated with single- and double-stranded
isolated calf thymus DNA.
Conclusions
As the parent compounds of a series of
halogenated pesticides, aldrin and
dieldrin are cytotoxic to mammalian cells
in vitro. The metabolic activation of aldrin
produces a product with greater DNA
damaging potential than the parent
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compound. Modification of the 6,7-
double bond of aldrin to the epoxide
(dieldrin) results in extensive damage to
mammalian cells in vitro relative to the
parent compound. However, reduction of
the 6,7-double bond greatly reduces the
mammalian cell cytotoxicity and DNA
damaging capabilities of the parent
compound. Incorporation of a 6-fluoro
group into the reduced molecule
produced an agent with minimal DNA-
damaging capabilities.
Chemical manipulation of the bridge
carbon to produce oxyaldrin and
oxydieldrin produced agents with DNA-
damaging capabilities. Studies utilizing
6,7-dihydroaldrin indicated that
mammalian cell cytotoxicity and DNA
damage could be reduced and a high level
of insect toxicity could still be retained.
The results of this study support the
feasibility of the rational design of
pesticides that will not induce genetic
damage resulting in mutagenesis,
carcinogenesis, and cytotoxicity. The
selection of a limited number of model
compounds with modified chemical
functional groups served as an effective
basis for identifying the probable sites of
reactivity for insecticidal and mammalian
cellular effects in the aldrin/dieldrin
class of halogenated pesticides. Thus, it is
possible to reducethedeleteriousgenetic
effects of many of these agents in
mammalian cell systems selectively
while maintaining a high level of insect
toxicity This research suggests that more
effective and environmentally safe
pesticides may be attainable through an
interdisciplinary approach combining
chemical, cellular, molecular and
entomological studies.
5. M. D'Ambrosia, N. J. Lewis, R. W. Hart, and W. J. Collins are with Ohio State
University, Columbus. OH 43210.
Stephen Nesnow is the EPA Project Officer (see below).
The complete report, entitled "Chlorinated Hydrocarbons: Insecticide Versus
Carcinogenic Action," (Order No. PB 83-181 578; Cost: $8.50, 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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United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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