Demographic, endocrine and behavioral responses to mirex in the South polar skua.

Population consequences of chronic exposure to multiple pollutants at low environmental doses remain speculative, because of the lack of appropriate long-term monitoring surveys. This study integrates proximate and ultimate aspects of persistent organic pollutants (POP) burden in free-living vertebrates, by coupling hormonal and behavioral endpoints, life-history traits, and population dynamics. Blood samples (N=70) were collected in South polar skuas during two breeding periods, in 2003 and 2005, and individuals were annually monitored until 2011. Multi-state mark recapture models were used to test the effects of POP levels on demographic traits. Survival rate and long-term breeding probability were not related to individual POP levels, whereas long-term breeding success significantly decreased with increasing blood levels of mirex, an organochlorine insecticide. At the proximate level, corticosterone (stress hormone) and prolactin (parental care hormone) levels were not linked to individual POP burden. Nest defense in 2005 was significantly less intensive in chick-rearing skuas bearing higher mirex levels, suggesting reproductive behavioral impairment. Matrix population models were then built to project the rate of population decline according to increasing mirex burden. Although mirex levels were 2.8 times higher in 2003 than in 2005, the population-level effect of mirex was only detected in 2005, the year of higher corticosterone levels. The combination of endocrine traits with demographic analysis thereby enables to provide new support of synergistic interactions between pollutants and stress levels on long-term breeding outputs and population dynamics.

Secretory function of ovarian cells and myometrial contractions in cow are affected by chlorinated insecticides (chlordane, heptachlor, mirex) in vitro.

The aim of the study was to investigate the effect of chlordane, heptachlor and mirex, on hormonal regulation of the force of myometrial contractions. Myometrial, endometrial, granulosa and luteal cells as well as strips of myometrium from non-pregnant cows were incubated with three insecticides at environmentally relevant doses (0.1, 1 or 10ng/ml). None of the insecticides affected the viability of studied cells. Chlordane stimulated, while heptachlor and mirex inhibited, secretion of testosterone and estradiol from granulosa cells as well as secretion of progesterone from luteal cells, respectively. Secretion of oxytocin (OT) from granulosa cells was increased after incubation with all studied insecticides. Only mirex stimulated OT secretion from luteal cells, while heptachlor inhibited this effect. None of them affected synthesis of OT in luteal cells and prostaglandins (PGF2 and PGE2) secretion from uterine cells, except PGE2 secretion from endometrial cells was decreased when the cells were incubated with 0.1ng/ml of chlordane. Basal and OT-stimulated myometrial contractions were increased by mirex and decreased by heptachlor. The data show that the insecticides altered secretory function of ovarian cells. Heptachlor and mirex affected also myometrial contractions in vitro, but uterine secretion of prostaglandins were not involved in the mechanism of that adverse effect of insecticides. The data indicate on potential of these insecticides to disturb fertilisation, blastocyst implantation or even the length of gestation.

Organochlorine pesticides and risk of endometriosis: findings from a population-based case-control study.

BACKGROUND: Endometriosis is considered an estrogen-dependent disease. Persistent environmental chemicals that exhibit hormonal properties, such as organochlorine pesticides (OCPs), may affect endometriosis risk. OBJECTIVE: We investigated endometriosis risk in relation to environmental exposure to OCPs. METHODS: We conducted the present analyses using data from the Women's Risk of Endometriosis (WREN) study, a population-based case-control study of endometriosis conducted among 18- to 49-year-old female enrollees of a large health care system in western Washington State. OCP concentrations were measured in sera from surgically confirmed endometriosis cases (n = 248) first diagnosed between 1996 and 2001 and from population-based controls (n = 538). We estimated odds ratios (OR) and 95% CIs using unconditional logistic regression, adjusting for age, reference date year, serum lipids, education, race/ethnicity, smoking, and alcohol intake. RESULTS: Our data suggested increased endometriosis risk associated with serum concentrations of ß-hexachlorocyclohexane (HCH) (third vs. lowest quartile: OR = 1.7; 95% CI: 1.0, 2.8; highest vs. lowest quartile OR = 1.3; 95% CI: 0.8, 2.4) and mirex (highest vs. lowest category: OR = 1.5; 95% CI: 1.0, 2.2). The association between serum ß-HCH concentrations and endometriosis was stronger in analyses restricting cases to those with ovarian endometriosis (third vs. lowest quartile: OR = 2.5; 95% CI: 1.5, 5.2; highest vs. lowest quartile: OR = 2.5; 95% CI: 1.1, 5.3). CONCLUSIONS: In our case-control study of women enrolled in a large health care system in the U.S. Pacific Northwest, serum concentrations of ß-HCH and mirex were positively associated with endometriosis. Extensive past use of environmentally persistent OCPs in the United States or present use in other countries may affect the health of reproductive-age women.

Characteristic molecular signature for early detection and prediction of persistent organic pollutants in rat liver.

Persistent organic pollutants (POPs) are degradation-resistant anthropogenic chemicals that accumulate in the food chain and in adipose tissue, and are among the most hazardous compounds ever synthesized. However, their toxic mechanisms are still undefined. To investigate whether characteristic molecular signatures can discriminate individual POP and provide prediction markers for the early detection of POPs exposure in an animal model, we performed transcriptomic analysis of rat liver tissues after exposure to POPs. The six different POPs (toxaphene, hexachlorobenzene, chlordane, mirex, dieldrin, and heptachlor) were administered to 11-week-old male Sprague-Dawley rats, and after 48 h of exposure, RNAs were extracted from liver tissues and subjected to rat whole genome expression microarrays. Early during exposure, conventional toxicological analysis including changes in the body and organ weight, histopathological examination, and blood biochemical analysis did not reflect any toxicant stresses. However, unsupervised gene expression analysis of rat liver tissues revealed in a characteristic molecular signature for each toxicant, and supervised analysis identified 2708 outlier genes that discerned the POPs exposure group from the vehicle-treated control. Combination analysis of two different multiclassifications suggested 384 genes as early detection markers for predicting each POP exposure with 100% accuracy. The data from large-scale gene expression analysis of a different POP exposure in rat model suggest that characteristic expression profiles exist in liver hepatic cells and multiclassification of POP-specific molecular signatures can discriminate each toxicant at an early exposure time. The use of these molecular markers may be more widely implemented in combination with more traditional techniques for assessment and prediction of toxicity exposure to POPs from an environmental aspect.

Exposure to persistent organic pollutants (POPs) and DNA damage as an indicator of environmental stress in fish of different feeding habits of Coatzacoalcos, Veracruz, Mexico.

The region of Coatzacoalcos, Veracruz hosts one of the largest and most important industrial areas of Mexico and Latin America. Industrial development and rapid population growth, have triggered a severe impact on aquatic ecosystems of the region. The aim of this study was to determine the levels of POPs in sediment and in muscle tissue of five fish species from different trophic levels in downstream residents of the Coatzacoalcos River, and their integration with DNA damage in the fish, evaluated with the comet assay in whole blood as a biological indicator of stress, in order to obtain a baseline of the ecological condition of the region. The compounds detected in sediment and in muscle tissue were hexachlorobenzene (HCB), α-, ß-, γ-hexachlorocyclohexane (HCH), dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), mirex and polychlorinated biphenyls (PCBs). Sediment concentrations of these pollutants (except for mirex) exceeded the values of protection provided by international guidelines, suggesting a potential risk to aquatic life in the region. DNA damage recorded in the fish species is evidence of exposure to a mix of genotoxic pollutants, which combined with exposure to POPs, reflects the degree of environmental stress of aquatic organisms in the region. The results of this study show the importance of determining the presence of contaminants in the environment, the bioaccumulation in tissues and their effects on exposed organisms, providing an integrated approach in assessing the health of aquatic ecosystems.

Exploring associations between serum levels of select organochlorines and thyroxine in a sample of New York state sportsmen: the New York State Angler Cohort Study.

This preliminary study investigated associations between environmental organochlorine compounds and thyroid function in a sample of 66 sportsmen selected from among participants in the New York State Angler Cohort Study. A cross-sectional design was employed with the primary goal of the analysis being the generation of specific testable hypotheses. Blood samples were analyzed for compounds based on a priori identified literature-cited evidence of thyroid disruption. These included hexachlorobenzene and polychlorinated biphenyl congeners 19, 28, 47, 118, 153, 169, 180, 183, and 187. Time of sample collection, serum triglycerides, cholesterol, high- and low-density lipoproteins, age, body mass index, and cigarette smoking were considered for each participant. Potential associations between organochlorine compounds and serum total thyroxine, controlling for potential confounders, were examined using multivariable linear regression models. The models reported consisted of all variates being entered ("full" model, R2=0.380, P=0.136) and stepwise selection of variates ("reduced" models, alpha=0.15) using the criterion of maximum partial correlation at each step. Several procedures were considered to address contaminant data below the limit of detection in the reduced models with no change in selected predictors. Hexachlorobenzene (beta=-0.113) and age (beta=0.007) were selected as predictors of serum T4 in the reduced models (R2=0.083, P=0.065). Power analysis suggested that by doubling the sample size the existing results would be statistically significant with a type I error of 0.05 and a power of 0.80. These findings are important in the design of a new specific study of thyroid function and environmental contaminants.

17beta-estradiol is a hormonal regulator of mirex tumor promotion sensitivity in mice.

Mirex, an organochlorine pesticide, is a potent non-phorbol ester tumor promoter in mouse skin. Previous studies have shown that female mice are 3 times more sensitive to mirex tumor promotion than male mice and that ovariectomized (OVX) female mice are resistant to mirex promotion, suggesting a role for ovarian hormones in mirex promotion. To determine whether the ovarian hormone 17-beta estradiol (E2) is responsible for the sensitivity of female mice to mirex promotion, female mice were initiated with DMBA; 2 weeks later groups of mice were OVX and implants, with or without E2, were surgically implanted subcutaneously. These mice were treated topically twice weekly with mirex for 26 weeks. E2 implanted OVX mice demonstrated high normal physiologic levels of serum E2 throughout the tumor promotion experiment. E2 implants restored by 80% the intact mirex-sensitive phenotype to the OVX mice. Consistent with a role for E2 and ERalpha and ERbeta, treatment of DMBA-initiated female mice with topical ICI 182,780, an estrogen-receptor antagonist, reduced mirex tumor multiplicity by 30%. However, in cells co-transfected with ERalpha or ERbeta and estrogen-responsive promoter reporter, mirex did not stimulate promoter reporter activity, suggesting that the promotion effect of mirex is downstream of ERalpha/beta. Finally, a tumor promotion study was conducted to determine whether E2 implants could increase the sensitivity of male mice to mirex promotion. E2 implants in male mice did increase sensitivity to mirex promotion; however, the implants did not produce the full female sensitivity to mirex tumor promotion. Collectively, these studies indicate that E2 is a major ovarian hormone responsible for mirex tumor promotion sensitivity in female mice.

Embryotoxicity of the pesticide mirex in vitro.

Mirex is a pesticide that is environmentally stable, accumulates in body tissues, and is embryo- and feto-toxic at high concentrations in vivo. This study is the first to evaluate the effects of mirex on organogenesis-stage embryos in vitro. Mouse embryos were exposed on gestation day 8.5 for 24 h in whole-embryo culture to mirex at 100, 200, or 400 microg/ml dissolved in xylene and compared with xylene-treated controls (1, 2, or 4 microl/ml, respectively) and untreated controls. Embryos were evaluated for malformations, somite number, total protein content, and visceral yolk sac circulation. Potential embryotoxic mechanisms were evaluated by using PCNA stain for cell proliferation and the TUNEL assay for apoptotic cell death. Mirex-exposed embryos demonstrated increased malformation rates and decreased total embryonic protein contents at > or =200 microg/ml mirex, and decreased somite numbers and VYS circulation at > or =100 microg/ml mirex, compared with xylene-treated controls. There was no difference in PCNA levels or TUNEL staining in mirex-treated embryos compared with xylene-treated controls or untreated controls. Thus, mirex is embryotoxic in vitro to early organogenesis stage mouse embryos at concentrations > or =100 microg/ml, but the effects do not appear to be mediated by changes in cell proliferation or apoptotic cell death.

Quantitative determination of endocrine- disrupting polychlorinated biphenyls and organochlorinated pesticides in human serum using gas chromatography with electron-capture detection and tandem mass spectrometry.

A sensitive, selective and reliable procedure was developed and validated to determine organochlorinated compounds, which have endocrine-disrupting effects, in human serum. Target compounds were selected between polychlorinated biphenyls and organochlorinated pesticides. Sample workup consisted of (1) extraction of serum with organic solvents, (2) clean-up of the organic extract using acid treatment with H(2)SO(4), (3) elution of the cleaned-up extract through a liquid column chromatographic system and (4) analysis of the fraction eluted by gas chromatography with electron capture detection (ECD) and tandem mass spectrometry (MS/MS) detection. Performance characteristics, such as linearity, sensitivity, precision, accuracy and recovery, of both chromatographic methods were studied. The proposed analytical methodology was applied to determine the target compounds in serum samples from women living in agricultural areas of Almería (Spain). The results show the advantage of MS/MS over ECD in the analysis of real human serum samples where matrix interferences can be confused with target pesticides.

The effect of photomirex on the in vitro perfused ovary of the rat.

Photomirex, a photodegradation product of the insecticide mirex, is an environmental contaminant that has been identified in Great Lakes fish, soil, and human adipose tissue. Because of the potential for human exposure, the present study was designed to investigate the short-term effects of photomirex on the in vitro perfused ovary of the rat. Adult Sprague-Dawley rat ovaries were isolated and perfused for a total of 6 h with Medium 199. Following a 2-h baseline period, 10(-4) M of photomirex was administered to the medium. Control ovaries received medium or DMSO (vehicle control). Significant effects of perfusion and chemical intervention were identified using lactate dehydrogenase enzyme, glucose utilization, lactate, pyruvate, and flow:pressure ratio as markers of toxicity (P < 0.05). Lactate:pyruvate ratio, glutathione, and oxygen consumption did not demonstrate significant effects. Post hoc tests showed that there were significant differences between the DMSO + photomirex group and the control group (M199) using lactate dehydrogenase as a marker of toxicity. Pyruvate concentration was also reduced significantly after perfusion with DMSO + photomirex compared to M199 only and DMSO only (P < 0.05). Histopathologic changes were not discernible by light microscopy. These results suggest that metabolic and respiratory processes of the ovary are acutely sensitive to perturbation with photomirex in the in vitro perfused rat ovary model.

Effects of photoisomers of cyclodiene insecticides on liver and microsomal cytochrome P450 in rats.

Threshold dosages of the photoisomers of cyclodiene insecticides, namely photochlordane, photodieldrin, and photoheptachlor, for the induction of hepatic microsomal cytochrome P450 (P450) and liver hypertrophy in male rats were at least one-quarter of those reported for corresponding parent cyclodienes. Maximum increase in total P450 concentration (30%) and demethylases activities (100%) was always respectively one-third or one-tenth of that reported for parent cyclodienes. The P450 isozymic form induced by photoheptachlor resembled that induced by pentobarbital (P4502B1) in its substrate specificity, spectral characteristics, and electrophoretic mobility. The induction of P450 was initially followed by hepatic hypertrophy. However, higher dosages of photoisomers caused wasting and lowered both the liver weight and the activity of aniline hydroxylase while those of mirex and endrin, which also caused wasting and lowered aniline hydroxylase activity, continued causing further hepatic hypertrophy.

Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha-ras genotype.

Mirex is a potent tumor promoter in 7,1 2-dimethylbenz[a]anthracene (DMBA)-initiated female CD-1 mouse skin. Like 12-O-tetradecanoylphorbol-13-acetate (TPA), mirex promotes papillomas that have a Ha-ras mutation; however, unlike TPA promotion, mirex promotion does not involve a general hyperplastic response. We used proliferating cell nuclear antigen (PCNA) and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemical staining to further examine the proliferative capacity of mirex. The numbers of PCNA- and BrdU-positive epidermal S-phase cells were highly concordant in all treatment groups. Unlike a single application of TPA, a single application of mirex had little or no effect on the number of S-phase epidermal cells, and chronic application of mirex to mouse skin produced only minimal increases in S-phase cells. Moreover, mirex did not significantly alter the growth of BALB/MK-2 keratinocytes in media containing either 0.05 or 1.2 mM Ca++. These results suggest that mirex may have highly specific effects on the proliferation of initiated cells and support the existence of a unique mirex mechanism and/or distinct population of mirex-promotable mutant Ha-ras epidermal cells. To begin to address this issue of a distinct population of mirex-promotable mutant Ha-ras cells, we conducted a tandem experiment in which DMBA-initiated mice were treated twice weekly with a maximal promoting dose of mirex. Then, when the number of papillomas reached a plateau, these same mice were treated twice weekly with a maximal promoting dose of TPA. Mice treated with mirex developed a maximum of 6.4 papillomas/mouse. These mice were then promoted with TPA, which produced 8.9 additional papillomas/mouse for a total of 15.3 papillomas/mouse. The maximum tumor yields from other groups of mice treated with only TPA or mirex were 9.8 and 7.3 papillomas/mouse, respectively. Therefore, under these tandem conditions, tumor yields were additive, indicating that there are at least two distinct populations of mutant Ha-ras cells: one promoted by mirex and the other by TPA.

Lack of effect of retinoic acid and fluocinolone acetonide on mirex tumor promotion indicates a novel mirex mechanism.

Mirex, a halogenated hydrocarbon, is a potent skin tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. In the present study retinoic acid (RA) and fluocinolone acetonide (FA), classical inhibitors of phorbol ester- and non-phorbol ester-type skin tumor promoters, were examined for their ability to inhibit mirex tumor promotion. Female CD-1 mice were initiated with 200 nmol DMBA and promoted with equipotent promoting doses of either 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) or 200 nmol mirex twice weekly for 25 weeks and RA (2(1 or 5 nmol), FA (0.5 or 2 nmol) or acetone were applied 30 min prior to each TPA or mirex dose. TPA-promoted papilloma formation was strongly inhibited by > 70% with both doses of RA and by > 90% with both doses of FA. In contrast, mirex-promoted papilloma formation was not inhibited by either dose of RA or 0.5 nmol FA and 2 nmol FA weakly inhibited mirex-promoted papillomas by only 32%. TPA- and mirex-promoted papillomas that were refractory to RA and FA demonstrated the same incidence of Ha-ras mutation as TPA- or mirex-promoted papillomas without RA and FA treatment, further indicating that the inhibitory activity of RA and FA is promoter-dependent and not solely dependent on mutant Ha-ras. FA (2 nmol) treatment completely abolished TPA-induced epidermal hyperplasia and proliferating cell nuclear antigen (PCNA) S phase-positive cells, however, FA had no inhibitory effect on the weak proliferative response induced by mirex. Collectively, these results indicate that the promotional activity of mirex, as well as its weak proliferative response, result from a distinct promoter mechanism and/or that mirex promotes a unique population of epidermal cells that are insensitive to FA and RA and cannot be distinguished by their mutant Ha-ras genotype.

Synergistic interaction between the non-phorbol ester-type promoter mirex and 12-O-tetradecanoylphorbol-13-acetate in mouse skin tumor promotion.

Mirex, an organochlorine pesticide and non-genotoxic rodent hepatocarcinogen, is also a potent non-phorbol ester-type promoter of mouse skin tumors. Mirex, unlike most other skin tumor promoters, is not a significant epidermal hyperplasiogen even at a maximally promoting dose (200 nmol). Experiments described here examined whether tumor promotion by mirex and 12-O-tetradecanoylphorbol-13-acetate (TPA) are mediated through different mechanisms as indicated by their additivity when co-applied to 7,12-dimethyl-benz[a]anthracene (DMBA, 200 nmol)-initiated female CD-1 mouse skin. Instead of the additive response of 14 plus 5 tumors/mouse predicted from mice promoted for 20 weeks (2x/week) with either mirex (200 nmol) or TPA (2 nmol) respectively, their co-application yielded 35 tumors/mouse. This synergy with TPA was specific to mirex since a structurally related compound, chlordecone (Kepone) was inactive. Mirex plus TPA-promoted papillomas contained a c-Ha-ras A182-->T mutation as frequently (13/14) as those promoted by mirex or TPA alone, suggesting that these DMBA-initiated/co-promoted papillomas were not atypical in this genotypic marker. Promotional synergy with mirex was only observed with a submaximal promoting dose of 2 nmol TPA; 5 or 8 nmol TPA plus mirex gave additive or less tumor multiplicities. This synergistic multiplicity with mirex plus 2 nmol TPA (35 tumors/mouse) approximated the sum of individual responses to 200 nmol mirex (14 tumors/mouse) and the maximally promoting dose of TPA (12 nmol), 24 tumors/mouse, suggesting that mirex potentiated the promotional activity of TPA, as well as promoted through a mirex-specific mechanism. Epidermal DNA synthesis induced by 2 nmol TPA was potentiated by mirex, further supporting a role for mirex in potentiation of epidermal TPA activity. Collectively, these studies suggest that mirex affects two possibly related responses: (i) promotion through a distinct mirex-specific mechanism, and (ii) potentiation of a mechanism mediating the promotional activity of TPA.

Minimal role of enhanced cell proliferation in skin tumor promotion by mirex: a nonphorbol ester-type promoter.

Mirex, a chlorinated hydrocarbon previously used as a systemic insecticide and flame retardant, is a nongenotoxic hepatocarcinogen in both rats and mice. In liver, mirex induced biochemical responses and hyperplasia characteristic of increased cell proliferation, which is consistent with its role as a liver tumor promoter. We have recently shown that mirex is a potent nonphorbol ester-type skin tumor promoter in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated mice. However, unlike its effect in liver, a single topical application of mirex to skin does not induce the acute biochemical responses, such as increased epidermal DNA synthesis and ornithine decarboxylase activity, indicative of increased cell proliferation. Multiple topical applications of mirex over a 1 month period induced only a minimal increase in the number of epidermal nucleated cell layers, which contrasts with definitive hyperplasia induced by a comparable tumor-promoting dose of 12-O-tetradecanoylphorbol-13-acetate (TPA). Collectively, these data indicated that mirex is promoting through a novel mechanism. Further evidence that mirex promotes tumors through a mechanism distinct from that of the prototypical skin tumor promoter, TPA, was obtained by examining the effect of their simultaneous co-treatment. The co-application of mirex and TPA yielded a tumor multiplicity greater than the sum of the responses of each promoter individually. In summary, our results demonstrate that mirex, a carcinogenic and hyperplastic agent in liver, is also a very effective tumor promoter in mouse skin, but suggest that mirex operates via a novel mechanism in skin that may involve only a minimal role for enhanced cell proliferation.

Characterization of skin tumor promotion by mirex: structure-activity relationships, sexual dimorphism and presence of Ha-ras mutation.

In the present study we have compared the tumor-promoting activity of the non-phorbol ester-type skin tumor promoter, mirex, a halogenated cycloalkane pesticide, to the following: (i) chlordane, a halogenated cycloalkane pesticide; (ii) 1,1-bis (4-chlorophenyl)-2,2,2-trichlorethane (DDT), a halogenated bridged aromatic pesticide; and (iii) kepone, a halogenated cycloalkane pesticide, which only differs from mirex by the substitution of two chlorine atoms with an oxygen atom. Topical application of 200 nmol mirex three times weekly for 20 weeks to 7,12-dimethylbenz[a]anthracene (DMBA)-initiated female mouse skin produced approximately 16 tumors/mouse with a 96% incidence of tumor bearing mice. Neither chlordane (2 mumol) or DDT (5 mumol) promoted tumors in DMBA-initiated mouse skin after three times weekly application for 20 weeks. Unexpectedly, DMBA-initiated mice treated with 250 nmol kepone three times weekly for 20 weeks did not develop any tumors, demonstrating that the replacement of two chlorine atoms by an oxygen atom results in loss of the skin tumor-promoting activity of mirex. To further characterize mirex-induced skin tumor promotion, male mice were initiated with a single topical application of 200 nmol DMBA and promoted topically three times weekly for 20 weeks with 200 nmol mirex. As compared to female mice, male mice demonstrated (i) 70% fewer tumors/mouse, (ii) decreased incidence of tumor bearing mice, (iii) increased time to first tumor and (iv) increased latency. To determine the role of ovarian hormones in the increased sensitivity of female mice, mice were initiated with DMBA, ovariectomized (OVX) 2 weeks later and then promoted with mirex. OVX mice exhibited 70% fewer tumors/mouse and a 40% decrease in incidence of tumor-bearing mice as compared to controls. Finally, > 90% of DMBA-initiated/mirex-promoted papillomas from male mice and female mice demonstrated a mutated Ha-ras gene with an A-->T transversion in the middle base of the 61st codon. Collectively, these data indicate that the tumor-promoting ability of mirex is highly structure specific, and ovarian hormones are a factor in the increased sensitivity of female mice to the skin tumor-promoting ability of mirex. Furthermore, mirex appears to clonally expand epidermal cells with a mutated Ha-ras oncogene.