Induction of chromosmal aberrations in the syrian hamster by insecticides tested in vivo

The insecticides demeton, dimetoat, dichlorovos, endosulfan, trichlorofon, carbaryl, lindane, methoxychlor, propoxur and malathion were examined for their ability to induce chromosomal aberrations in the bone marrow cells of the Syrian hamster (Mesocricetus auratus) treated in vivo. Mutagenicity of commercial preparations was examined at four doses: LD₅₀; 1/2, 1/5 and 1/10 LD₅₀. The positive control was an IP injection of cyclophosphamide to hamsters at a dose of 40 mg/kg body wt. Demeton, dichlorovos and endosulfan gave positive results. Malathion, dimethoate and the mixture of methoxychlor and propoxur were weakly clastogenic; at some doses these compounds induced statistically significant increases in the number of aberrations. Trichlorfon and the mixture of carbaryl and lindane were negative in this test.This work was supported by the Polish Academy of Sciences within the projet 09.7.3.4.3     

David Kupfer,Ph.D. A Mentor and a Scientist

I worked with the late Dr. David Kupfer for nearly nine years at the Worcester Foundation/University of Massachusetts Medical School, Worcester, MA. I was involved in the metabolism of methoxychlor and tamoxifen, the areas of research close to David's heart. We demonstrated the metabolic pathways of these compounds in rats and humans, and the covalent binding to microsomal proteins that could result in long-term toxic manifestations. I learned a lot from David, who was a mentor and friend/colleague. His death has left a void in my heart and he will be sorely missed.     

David Kupfer: A Career Retrospective

David Kupfer's research career spanned 50 years and he authored or co-authored over 160 papers and book chapters. Although best known for his work centering on cytochrome P450 metabolism of prostaglandins, steroids, and proestrogenic compounds, David's research also contributed key advances in the areas of P450 induction and catalytic mechanism, breast cancer therapy, and analytical methodology. His research is reviewed here.     

The methoxychlor metabolite, HPTE, inhibits rat luteal cell progesterone production

The methoxychlor metabolite, HPTE, was shown to inhibit P450-cholesterol side-chain cleavage (P450scc) activity resulting in decreased progesterone production by cultured ovarian follicular cells in previous studies. It is not known whether HPTE has any effect on progesterone formation by the corpus luteum.

Results

Exposure to 100 nM HPTE reduced progesterone production by luteal cells with progressive declines to <22% of control at 500 nM HPTE. Similarly, HPTE progressively inhibited progesterone formation and P450scc catalytic activity of hCG- or 8 Br-cAMP-stimulated luteal cells. However, HPTE did not alter mRNA and protein levels of P450scc. Compounds acting as estrogen (17β-estradiol, bisphenol-A or octylphenol), antiestrogen (ICI) or antiandrogen (monobutyl phthalate, flutamide or M-2) added alone to luteal cells did not mimic the action of HPTE on progesterone and P450scc activity. These results suggest that HPTE directly inhibits P450scc catalytic activity resulting in reduced progesterone formation, and this action was not mediated through estrogen or androgen receptors.     

Methoxychlor inhibits growth of antral follicles by altering cell cycle regulators

Methoxychlor (MXC) reduces fertility in female rodents, decreases antral follicle numbers, and increases atresia through oxidative stress pathways. MXC also inhibits antral follicle growth in vitro. The mechanism by which MXC inhibits growth of follicles is unknown. The growth of follicles is controlled, in part, by cell cycle regulators. Thus, we tested the hypothesis that MXC inhibits follicle growth by reducing the levels of selected cell cycle regulators. Further, we tested whether co-treatment with an antioxidant, N-acetyl cysteine (NAC), prevents the MXC-induced reduction in cell cycle regulators. For in vivo studies, adult cycling CD-1 mice were dosed with MXC or vehicle for 20 days. Treated ovaries were subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA) staining. For in vitro studies, antral follicles isolated from adult cycling CD-1 mouse ovaries were cultured with vehicle, MXC, and/or NAC for 48, 72 and 96 h. Levels of cyclin D2 (Ccnd2) and cyclin dependent kinase 4 (Cdk4) were measured using in vivo and in vitro samples. The results indicate that MXC decreased PCNA staining, and Ccnd2 and Cdk4 levels compared to controls. NAC co-treatment restored follicle growth and expression of Ccnd2 and Cdk4. Collectively, these data indicate that MXC exposure reduces the levels of Ccnd2 and Cdk4 in follicles, and that protection from oxidative stress restores Ccnd2 and Cdk4 levels. Therefore, MXC-induced oxidative stress may decrease the levels of cell cycle regulators, which in turn, results in inhibition of the growth of antral follicles.     

Interstitial cell carcinomas of the testis in Balb/C male mice ingesting methoxychlor

Summary Balb/c and C3H strains male and female mice ingested 750 ppm methoxychlor or 100 ppm DDT in the diet for 2 years. Balb/c strain male mice ingesting methoxychlor developed a highly significant incidence of interstitial cell carcinomas of the testis. Balb/c strain male mice ingesting DDT and C3H strain male mice receiving methoxychlor or DDT did not have testicular tumors. The carcinomas of the testis varied from well-differentiated to poorly differentiated and undifferentiated and were capable of metastasis. Carcinomas of the testis have been described in Balb/c strain male mice, but not C3H, given estrogens. The carcinogenicity for testis of Balb/c strain male mice is most likely related to the estrogenic activity of methoxychlor.The materials relevant to this study, including tissue slides, were submitted to the Environmental Defense Fund and to the author by the Food and Drug Administration, Washington, D.C.     

Methoxychlor causes mitochondrial dysfunction and oxidative damage in the mouse ovary

Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by causing ovarian atrophy, persistent estrous cyclicity, and antral follicle atresia (apoptotic cell death). Oxidative damage resulting from reactive oxygen species (ROS) generation has been demonstrated to lead to toxicant-induced cell death. Thus, this work tested the hypothesis that MXC causes oxidative damage to the mouse ovary and affects mitochondrial respiration in a manner that stimulates ROS production. For the in vitro experiments, mitochondria were collected from adult cycling mouse ovaries, treated with vehicle (dimethyl sulfoxide; DMSO) or MXC, and subjected to polarographic measurements of respiration. For the in vivo experiments, adult cycling CD-1 mice were dosed with either vehicle (sesame oil) or MXC for 20 days. After treatment, ovarian mitochondria were isolated and subjected to measurements of respiration and fluorimetric measurements of H2O2 production. Some ovaries were also fixed and processed for immunohistochemistry using antibodies for ROS production markers: nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHG). Ovaries from in vivo experiments were also used to measure the mRNA expression and activity of antioxidants such as Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT). The results indicate that MXC significantly impairs mitochondrial respiration, increases production of H2O2, causes more staining for nitrotyrosine and 8-OHG in antral follicles, and decreases the expression and activity of SOD1, GPX, and CAT as compared to controls. Collectively, these data indicate that MXC inhibits mitochondrial respiration, causes ROS production, and decreases antioxidant expression and activity in the ovary, specifically in the antral follicles. Therefore, it is possible that MXC causes atresia of ovarian antral follicles by inducing oxidative stress through mitochondrial production of ROS.     

Pregnenolone co-treatment partially restores steroidogenesis,but does not prevent growth inhibition and increased atresia in mouse ovarian antral follicles treated with mono-hydroxy methoxychlor

Mono-hydroxy methoxychlor (mono-OH MXC) is a metabolite of the pesticide, methoxychlor (MXC). Although MXC is known to decrease antral follicle numbers, and increase follicle death in rodents, not much is known about the ovarian effects of mono-OH MXC. Previous studies indicate that mono-OH MXC inhibits mouse antral follicle growth, increases follicle death, and inhibits steroidogenesis in vitro. Further, previous studies indicate that CYP11A1 expression and production of progesterone (P₄) may be the early targets of mono-OH MXC in the steroidogenic pathway. Thus, this study tested whether supplementing pregnenolone, the precursor of progesterone and the substrate for HSD3B, would prevent decreased steroidogenesis, inhibited follicle growth, and increased follicle atresia in mono-OH MXC-treated follicles. Mouse antral follicles were exposed to vehicle (dimethylsulfoxide), mono-OH MXC (10 μg/mL), pregnenolone (1 μg/mL), or mono-OH MXC and pregnenolone together for 96 h. Levels of P₄, androstenedione (A), testosterone (T), estrone (E₁), and 17β-estradiol (E₂) in media were determined, and follicles were processed for histological evaluation of atresia. Pregnenolone treatment alone stimulated production of all steroid hormones except E₂. Mono-OH MXC-treated follicles had decreased sex steroids, but when given pregnenolone, produced levels of P₄, A, T, and E₁ that were comparable to those in vehicle-treated follicles. Pregnenolone treatment did not prevent growth inhibition and increased atresia in mono-OH MXC-treated follicles. Collectively, these data support the idea that the most upstream effect of mono-OH MXC on steroidogenesis is by reducing the availability of pregnenolone, and that adding pregnenolone may not be sufficient to prevent inhibited follicle growth and survival.     

The Use of a Whole Animal Biophotonic Model as a Screen for the Angiogenic Potential of Estrogenic Compounds

Background: Vascular endothelial growth factor (VEGF) is essential for normal vascular growth and development during wound repair. VEGF is estrogen responsive and capable of regulating its own receptor, vascular endothelial growth factor receptor-2 (VEGFR-2). Several agricultural pesticides (e.g., methoxychlor) have estrogenic potential that can initiate inappropriate physiological responses in estrogenic-sensitive tissues following exposure in vivo. Thus, the current study was designed to determine whether the VEGFR-2-Luciferase (Luc) reporter transgenic mouse is a useful model for evaluating estrogenic tendencies of methoxychlor by monitoring wound healing via VEGFR-2-mediated gene expression using bioluminescence and real-time imaging technology.Results: VEGFR-2-Luc gene activity peaked by d 7 (P<0.001) in all groups but was not different (P>0.05) between control and estrogen/methoxychlor exposed mice.Conclusions: Changes in VEGFR-2-Luc gene activity associated with the dermal wound healing process were able to be measured via photonic emission. The increase in vasculature recruitment and formation is paralleled by the increase of VEGFR-2-Luc activity with a peak on day 7. However, estrogen/methoxychlor did not significantly alter wound healing mediated VEGFR-2-Luc gene expression patterns compared to controls. This suggests that the VEGFR-2-Luc transgenic mouse wound model tested in this study may not be optimal for use as a screen for the angiogenic potential of estrogenic compounds.     

有机氯农药干扰妇女免疫、内分泌所致子宫内膜异位症的研究

目的通过对农村接触过甲氧滴滴涕(MXC)且患有子宫内膜异位症(EMs)的妇女进行MXC干扰妇女免疫、内分泌所致子宫内膜异位症的临床对照研究,为有机氯农药诱发子宫内膜异位症提供科学的证据。方法接触过MXC的EMs患者50例为观察组,未接触过MXC的EMs患者50例为对照组,另选30例正常妇女做参考值。采用气相色谱仪检测血清MXC,酶标法检测性激素促卵泡激素(FSH)、黄体生成激素(LH)、雌二醇(E2)、孕酮(P)水平、免疫因子白介素6(IL-6)、白介素8(IL-8)和肿瘤坏死因子α(TNF-α)。观察MXC与性激素的性惯性相关性。结果两组性激素分别与正常组比较,观察组Ⅰ~Ⅱ期、Ⅲ~Ⅳ期EMs患者FSH、LH、E2、P及对照组Ⅰ~Ⅱ期EMs患者FSH,Ⅲ~Ⅳ期FSH、E2、P有统计意义(P<0.05,P<0.01,P<0.001)。观察组与对照组比较,FSH、LH、E2、P均有统计学差异(P<0.05,P<0.001)。两组免疫因子与正常组比较,对照组Ⅰ~Ⅱ期、Ⅲ~Ⅳ期EMs患者IL-6、IL-8、TNF-α有统计学差异(P<0.05,P<0.01,P<0.001)。MXC与性激素相关性分析结果显示,MXC低、中、高残留量与FSH呈正相关,与E2呈负相关,有统计学差异(P<0.05)。结论 MXC与FSH、LH、E2、P有一定的相关性,MXC主要是通过影响性激素的分泌引发子宫内膜异位症。