Dysregulated estrogen receptor signaling in the hypothalamic-pituitary-ovarian axis leads to ovarian epithelial tumorigenesis in mice.

The etiology of ovarian epithelial cancer is poorly understood, mainly due to the lack of an appropriate experimental model for studying the onset and progression of this disease. We have created a mutant mouse model in which aberrant estrogen receptor alpha (ERα) signaling in the hypothalamic-pituitary-ovarian axis leads to ovarian epithelial tumorigenesis. In these mice, termed ERαd/d, the ERα gene was conditionally deleted in the anterior pituitary, but remained intact in the hypothalamus and the ovary. The loss of negative-feedback regulation by estrogen (E) at the level of the pituitary led to increased production of luteinizing hormone (LH) by this tissue. Hyperstimulation of the ovarian cells by LH resulted in elevated steroidogenesis, producing high circulating levels of steroid hormones, including E. The ERαd/d mice exhibited formation of palpable ovarian epithelial tumors starting at 5 months of age with 100% penetrance. By 15 months of age, 80% of ERαd/d mice die. Besides proliferating epithelial cells, these tumors also contained an expanded population of luteinized stromal cells, which acquire the ability to express P450 aromatase and synthesize E locally. In response to the elevated levels of E, the ERα signaling was accentuated in the ovarian epithelial cells of ERαd/d mice, triggering increased ERα-dependent gene expression, abnormal cell proliferation, and tumorigenesis. Consistent with these findings, treatment of ERαd/d mice with letrozole, an aromatase inhibitor, markedly reduced circulating E and ovarian tumor volume. We have, therefore, developed a unique animal model, which serves as a useful tool for exploring the involvement of E-dependent signaling pathways in ovarian epithelial tumorigenesis.

Does dietary copper supplementation enhance or diminish PCB126 toxicity in the rodent liver?

Copper is essential for the function of the mitochondrial electron transport chain and several antioxidant proteins. However, in its free form copper can participate in Fenton-like reactions that produce reactive hydroxyl radicals. Aryl-hydrocarbon receptor (AhR) agonists, including the most potent polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), increase copper levels in rodent livers. This is accompanied by biochemical and toxic changes. To assess the involvement of copper in PCB toxicity, male Sprague-Dawley rats were fed an AIN-93G diet with differing dietary copper levels: low (2 ppm), adequate (6 ppm), and high (10 ppm). After three weeks, rats from each group were given a single ip injection of corn oil (control), 1, or 5 µmol/kg body weight PCB126. Two weeks following injections, biochemical and morphological markers of hepatic toxicity, trace metal status, and hepatic gene expression of metalloproteins were evaluated. Increasing dietary copper was associated with elevated tissue levels of copper and ceruloplasmin. In the livers of PCB126-treated rats, the hallmark signs of AhR activation were present, including increased cytochrome P450 and lipid levels and decreased glutathione. In addition, a doubling of hepatic copper levels was seen, and overall metal homeostasis was disturbed, resulting in decreased hepatic selenium, manganese, zinc, and iron. Expression of key metalloproteins was either decreased (cytochrome c oxidase), unchanged (ceruloplasmin and CuZnSOD), or increased (tyrosinase and metallothioneins 1 and 2) with exposure to PCB126. Increases in metallothionein may contribute/reflect the increased copper seen. Alterations in dietary copper did not amplify or abrogate the hepatic toxicity of PCB126. PCB126 toxicity, i.e., oxidative stress and steatosis, is clearly associated with disturbed metal homeostasis. Understanding the mechanisms of this disturbance may provide tools to prevent liver toxicity by other AhR agonists.

Dietary selenium as a modulator of PCB 126-induced hepatotoxicity in male Sprague-Dawley rats.

Homeostasis of selenium (Se), a critical antioxidant incorporated into amino acids and enzymes, is disrupted by exposure to aryl hydrocarbon receptor (AhR) agonists. Here we examined the importance of dietary Se in preventing the toxicity of the most toxic polychlorinated biphenyl congener, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), a potent AhR agonist. Male Sprague-Dawley rats were fed a modified AIN-93 diet with differing dietary Se levels (0.02, 0.2, and 2 ppm). Following 3 weeks of acclimatization, rats from each dietary group were given a single ip injection of corn oil (vehicle), 0.2, 1, or 5 µmol/kg body weight PCB 126, followed 2 weeks later by euthanasia. PCB exposure caused dose-dependent increases in liver weight and at the highest PCB 126 dose decreases in whole body weight gains. Hepatic cytochrome P-450 (CYP1A1) activity was significantly increased even at the lowest dose of PCB 126, indicating potent AhR activation. PCB exposure diminished hepatic Se levels in a dose-dependent manner, and this was accompanied by diminished Se-dependent glutathione peroxidase activity. Both these effects were partially mitigated by Se supplementation. Conversely, thioredoxin (Trx) reductase activity and Trx oxidation state, although significantly diminished in the lowest dietary Se groups, were not affected by PCB exposure. In addition, PCB 126-induced changes in hepatic copper, iron, manganese, and zinc were observed. These results demonstrate that supplemental dietary Se was not able to completely prevent the toxicity caused by PCB 126 but was able to increase moderately the levels of several key antioxidants, thereby maintaining them roughly at normal levels.

Acute toxicity of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in male Sprague-Dawley rats: effects on hepatic oxidative stress, glutathione and metals status.

Although polychlorinated biphenyl (PCBs) production, and new uses for PCBs, was halted in the 1970s in the United States, PCBs continue to be used in closed systems and persist in the environment, accumulating in fatty tissues. PCBs are efficacious inducers of drug metabolism and may increase oxidative events and alter many other biochemical and morphologic parameters within cells and tissues. The goal of the present study was to evaluate the effects of a single, very low dose of PCB 126 (3,3',4,4',5-pentachlorobiphenyl), a coplanar, dioxin-like PCB congener and aryl hydrocarbon receptor (AhR) agonist, on redox status, metals homeostasis, antioxidant enzymes, and cellular morphology. To examine these parameters, male Sprague-Dawley rats were fed a purified AIN-93 basal diet containing 0.2 ppm selenium for two weeks, then administered a single i.p. injection of corn oil (5 ml/kg body weight) or 1µmol PCB 126/kg body weight (326µg/kg body weight) in corn oil. Rats were maintained on the diet for an additional two weeks before being euthanized. This dose of PCB 126 did not alter feed intake or growth, but significantly increased liver weight (42%) and hepatic microsomal cytochrome P-450 (CYP1A) enzyme activities (10-40-fold increase). Hepatic zinc, selenium, and glutathione levels were significantly decreased 15%, 30%, and 20%, respectively, by PCB 126. These changes were accompanied by a 60% decrease in selenium-dependent glutathione peroxidase activity. In contrast, hepatic copper levels were increased 40% by PCB 126. PCB 126-induced pathology was characterized by hepatocellular hypertrophy and mild steatosis in the liver and a mild decrease in cortical T-cells in the thymus. This controlled study in rats fed a purified diet shows that even a single, very low dose of PCB 126 that did not alter feed intake or growth, significantly perturbed redox and metals homeostasis and antioxidant and enzyme levels in rodent liver.

Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice.

Delta-6 desaturase-null mice ((-/-)) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The (-/-) males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type ((+/+)) and (-/-) males at weaning until 16 weeks of age (n = 3-5). A breeding success rate of DHA-supplemented (-/-) was comparable to (+/+). DHA-fed (-/-) showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed (-/-), but DPAn6 remained depleted. In AA-fed (-/-), AA was restored at the (+/+) level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of (+/+) and DHA-fed (-/-), whereas it diminished in (-/-) and AA-fed (-/-), suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (-/-), which enabled us to study HUFA deficiency without depleting their precursors. In -/-, no in vivo AA synthesis was detected after administration of [U-(13)C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the -/- developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The -/- also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male -/- lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in -/- declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in -/-. DPAn-6 was absent in -/- brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (-/-) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.