Evidence for the involvement of the fatty acid and peroxisomal beta-oxidation pathways in the inhibition by dehydroepiandrosterone (DHEA) and induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benz(a)anthracene (BA) of cytochrome P4501B1 (CYP1B1) in mouse embryo fibroblasts (C3H10T1/2 cells).

Treatment of intact C3H10T1/2 cells or microsomes therefrom with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzanthracene (BA) enhanced CYP1B1 activity and CYP1B1 expression as revealed by elevations of CYP1B1-catalyzed DMBA metabolism, CYP1B1 apoprotein level and CYP1B1 gene expression. One hundred microM DHEA caused an 80-90% inhibition of cellular DMBA metabolism without inflicting cell death. Cytosolic glucose-6-phosphate dehydrogenase (G6PDH) was also inhibited in DHEA-treated cells, presumably due to the inhibition of NADP reduction. In contrast, neither DMBA metabolism nor CYP1B1 apoprotein was inhibited by DHEA in the microsomes isolated from these cells. DHEA (100 microM), TCDD (10 nM) and BA (10 microM) stimulated the activities and increased the apoprotein levels of two peroxisomal enzymes, namely, acyl CoA oxidase (ACOX) and acyl CoA hydrolase (ACH2) and also induced the expression of CYP1B1 and ACOX genes. Cytosolic fatty acyl-CoA beta-oxidation was also stimulated by DHEA, TCDD and BA. In corroboratory experiments, it was found that concomitant with the stimulation of the activity of a key enzyme regulator of fatty acid homeostasis, namely, glycerol-3-phosphate dehydrogenase (G3PDH), these agents enhanced arachidonic acid (AA) metabolism as judged by the release of [3H] from AA into the culture medium. Collectively, these data suggest that DHEA mediates the regulation of CYP1B1 and inhibits BA and TCDD-induced CYP1B1-catalyzed carcinogen (DMBA) activation in 10T1/2 cells through metabolic interactions that involve the activation of the peroxisomal and fatty acid beta-oxidation signaling pathways. These results also present evidence for the first time, for the possible peroxisomal effects of TCDD and BA which are similar to those of DHEA in this mouse embryo fibroblast cell line.

Regulation of cytochrome P4501B1 (CYP1B1) in mouse embryo fibroblast (C3H10T1/2) cells by protein kinase C (PKC).

The effects of co-treatment of C3H10T1/2 (10T1/2) cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of the novel cytochrome P4501B1 (CYP1B1) were investigated. As monitored by CYP1B1-catalyzed 7,12-dimethylbenzanthracene (DMBA) metabolism, TPA suppressed basal and TCDD-induced DMBA metabolism in a concentration-dependent manner, with a maximum inhibitory concentration of 100 nM. The suppression of CYP1B1 catalytic activity occurred at two time points during which protein kinase C (PKC) was activated and down-regulated in these cells as judged by analyses of cellular PKC content and PKC-inhibitor (chelerythrine chloride)-influenced suppression of CYP1B1 catalytic activity. Experiments in which TCDD and benzanthracene (BA)-induced DMBA metabolism were monitored in PKCbeta1-overexpressing 10T1/2 cells revealed that the suppression of CYP1B1 activity is a consequence of cellular PKC elevation. This suppression phenomenon could be accounted for by PKC-mediated suppression of TCDD-induced CYP1B1 mRNA and apoprotein and of nuclear translocation of the Ah-receptor. In contrast, the mitogen-activated protein kinase (MAPK) proteins ERKs 1 and 2 were stimulated by TCDD under conditions in which PKC was activated. Collectively, our results suggest that PKC participates in the regulation of CYP1B1 in 10T1/2 cells, positively by directly suppressing the Ah-receptor signaling pathway, followed by an indirect or negative activation of the MAPK signaling pathway.

The regulation by gender, strain, dose, and feeding status of the induction of multiple forms of cytochrome P450 isozymes in rat hepatic microsomes by 2,4,5,2',4',5'-hexachlorobiphenyl.

2,4,5,2',4',5'-hexachlorobiphenyl (HCB) induces hepatic microsomal cytochromes P450 with a similar selectivity for responsive genes to phenobarbital (PB). CYP2Bl, CYP2B2, CYP2C6, CYP3Al, and CYP2Al each showed large strain differences in induction by HCB Fisher F344 >> Wistar Furth (WF) that were much more evident in female rats, paralleling previous observations with PB. These five P450s and epoxide hydrolase were, however, induced more effectively by HCB than by PB and strain differences were even larger. With HCB, strain differences in male rats were much more apparent than with PB. This change was not due to the greater HCB induction since a 2-fold lower induction was maintained even with a 10-fold lower dose of HCB. The sex and strain differences were seen both by immunoblot analysis and by form-selective enzyme activity assays. induction of CYP2B1, CYP2B2, and CYP3A1 by HCB was decreased 3-fold when starvation during the final 24 hr was replaced by continuous feeding. This effect was similar in each strain and therefore independent of the regulatory processes associated with the differential suppression of induction in WF rats. This modulation of induction by feeding was also seen with PB which caused only a 30% lowering of induction in continuously fed F344 rats. A 52-kDa microsomal protein (p52) was prominently induced by both HCB and PB after starvation, while minor induction of a 50-kDa microsomal protein (p50) also occurred after the same treatment. Furthermore, a 100-kDa microsomal protein (p100) was induced by HCB but not by PB and only in rats that were continuously fed. These results suggest that the induction of multiple forms of P450 following HCB treatment functions through the same PB-stimulated pathway that shows a strain-dependent endocrine (GH/T3/testosterone)-sensitive suppression mechanism. The induction of p5O, p52, and plOO by HCB suggests the presence of at least two additional hepatic response mechanisms for HCB.

Biochemical assessment of the genotoxicity of the in vitro interaction between chlordecone and carbon tetrachloride in rat hepatocytes.

The genotoxic potential of the administration of carbon tetrachloride alone or carbon tetrachloride to chlordecone-pretreated rats was investigated using an in vivo-in vitro animal model and a battery of biochemical assays to measure DNA repair in rat hepatocytes. Whereas carbon tetrachloride alone was not genotoxic, chlordecone or chlordecone in combination with carbon tetrachloride was genotoxic. The need for further investigation into the mechanism underlying the interaction between chlordecone and carbon tetrachloride is indicated strongly by the results of the present study.

Effects of steroids on the synthesis and metabolism of phosphatidylethanol in phorbol ester-activated lymphocytes.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate activates the phospholipase D pathway in bovine lymphocytes, leading to a synthesis of phosphatidylethanol (PEt) from exogenous alcohol. Concomitant treatment of the cells with 10(-8) M etiocholanolone, dehydroepiandrosterone or 16 alpha-bromo-epiandrosterone results in the production of phosphatidylethanols that carry metabolically altered forms of arachidonic acid at position 2. The observed steroid response appears to be mediated by a receptor mechanism in that it depends on the synthesis of new RNA and protein. The spectrum of steroids producing the response suggests that the postulated receptor system may be distinct from the well-studied glucocorticoid, progesterone, estrogen and androgen specific receptors. The possible relevance of these new metabolites of ethanol to the problem of alcoholism in humans and to the field of carcinogenesis in general is discussed.

12-O-tetradecanoylphorbol-13-acetate activates the synthesis of phosphatidylethanol in animal cells exposed to ethanol.

Tumor-promoting phorbol esters acutely activate a pathway in lymphocytes leading to the synthesis and accumulation of phosphatidylethanol, using exogenous ethanol as a precursor. This product is a representative of a unique class of acidic glycerophospholipids in which the head group is a primary alcohol. The formation of this lipid, in response to different phorbol ester derivatives, correlates with their activity as tumor promoters and inducers of growth changes in a variety of animal cells. Since phosphatidylethanol represents an unusual metabolite of ethanol, it is proposed that studies of its synthesis and biological functions may also provide new perspectives on the biology of alcohol addiction as well as the role of this biological pathway in tumor promotion.

Effect of aflatoxicosis on acetylcholinesterase activity in the brain and adenohypophysis of the male rat.

The effects of 7 or 42 48-hourly i.p. injections of 20 micrograms aflatoxin B1 (AFB) on the acetylcholinesterase activity (AChE) in 8 brain areas and the adenohypophysis of the adult male rat were studied. Aflatoxicosis increased adenohypophysial AChE in direct proportion to the duration of intoxication, which also altered the distribution of AChE in the rat brain. With acute treatment, AChE was depressed in the cerebellum and hippocampus while in the chronically dosed rats AChE was drastically elevated in the mesencephalon and amygdala. Results suggest that AFB changes the ACh turnover and hence the cholinergic transmission in the brain and adenohypophysis. This may result in behavioural deficits and/or performance decrements via a disturbance of the hypothalamo-hypophysial axis.

Zinc, copper, manganese and iron in rat organs after the administration and withdrawal of aflatoxin B1.

The in vivo levels of zinc, copper, manganese and iron were determined in the testis, kidney and three brain regions (cerebellum, cerebrum and medulla oblongata) from rats intraperitoneally injected with 20 micrograms per kg body weight of aflatoxin B1 for 6 weeks. Similar investigations were made after a recovery period of 6 weeks. The results obtained were compared with those of pair-fed control rats. The aflatoxin B1-induced decrease in testicular zinc was not reversed after the recovery of the rats. On the contrary, the significantly increased zinc concentration in the other organs following aflatoxin B1 exposure, reverted to the control level, as judged by significant levels. Only in the kidney was there accumulation of copper, even after the cessation of aflatoxin B1 administration. The marked increase in the manganese concentration of all the organs following repeated toxin administration, persisted only in the brain regions after the recovery. Aflatoxin B1 treatment significantly decreased the iron concentration in the testis, kidney, cerebellum and cerebrum. Recovery did not alter this trend. Iron concentration in the medulla oblongata was, however, not significantly changed in the presence of aflatoxin B1. The present data suggest that the repeated administration of aflatoxin B1 to rats could result in either a permanent or temporary re-distribution of these elements in the organs examined.

The neurotoxicity of aflatoxin B1 in the rat.

The effects of repeated intraperitoneal administration of aflatoxin B1 on the peripheral and central nervous systems of rats were investigated. Biochemical markers of neurotoxicity were monitored in nervous tissues following aflatoxin B1 dosage and after the cessation of aflatoxin B1 administration. Aflatoxin B1 increased the activities of beta-glucuronidase and beta-galactosidase in the central and peripheral nervous systems. Repeated exposure of rats to aflatoxin B1 also activated Na+ K+-ATPase and inhibited Mg2+-ATPase. Nervous tissue levels of DNA and total protein increased while the concentrations of RNA and phospholipid were depressed by aflatoxin B1. The alterations in these parameters were specific for each of the tissues examined during the recovery of the rats. The findings indicate that the repeated administration of aflatoxin B1 to rats results in degeneration in the central and peripheral nervous systems that may be related to the overt toxicity observed following aflatoxin administration.

Niridazole-induced biochemical changes in the rat testis.

A high dose of niridazole administered intraperitoneally (i.p.) to male rats over a period of 9 weeks induced alterations in the concentrations and activities of some important testicular constituents and enzymes, respectively. The concentrations of total and free cholesterol were higher than those of control animals but the concentration of esterified cholesterol was significantly lower than that of the control rats. Zinc concentration in the testis and prostate gland of niridazole-treated animals was significantly lower than in the control animals. Decreases in the activities of the following enzymes occurred in the testes of niridazole-treated rats: lactate and sorbitol dehydrogenases, aspartate and alanine aminotransferases and alkaline phosphatase. By contrast, there were increases in the activities of testicular malate dehydrogenase and prostate acid phosphatase of rats given niridazole.

Effects of kola-nut extract administration on the liver, kidney, brain, testis and some serum constituents of the rat.

Kola-nut extract induced a number of overt neurotoxicological signs in male albino rats. A decrease in the total body weight and an increase in the absolute weights of the liver, kidney, brain and testis were observed after 18 weeks oral administration of kola-nut extract to the rats. Total protein, RNA and DNA of these organs were significantly depressed. The activity of beta-glucuronidase and beta-galactosidase was induced only in the kidney, brain and testis of treated animals. While the levels of serum phosphomonoesterases and total cholesterol were significantly enhanced due to kola-nut intake, serum total and conjugated bilirubin levels were significantly decreased.

Effects of dimethylnitrosamine on some actions of testosterone.

Using homogenates of mouse kidney and testes, the activities of the enzymes, beta-glucuronidase and beta-galactosidase, were studied as markers of androgen action. The results obtained differed between testes and kidney homogenates. Dimethylnitrosamine (DMN) may cause a competitive inhibition of the anabolic action of testosterone in kidney homogenates but this was not evident from the results obtained with testes homogenates.

Sperm production rates, sperm physiology and fertility in rats chronically treated with sublethal doses of aflatoxin B1.

Rats chronically treated with sublethal doses of aflatoxin B1 suffered severe testicular degeneration and impaired spermatocytogenesis with a concomitant drop in the rate and efficiency of spermatozoal production. Although the major part of sperm maturation was disturbed, there was no noticeable reduction in the viability of spermatozoa in storage, hence a fairly high degree of fertilisation occurred. That the treatment made the uterine environment less compatible with normal foetal development was demonstrated by the smaller litter size and higher embryo mortality.

The effects of aflatoxin B1 on some testicular and kidney enzyme activity in rat.

The effects of chronic intra-peritoneal administration of aflatoxin B1 on the activity of alkaline and acid phosphatases; glutamic oxaloacetate (GOT) and pyruvate transaminases (GPT); 5'-nucleotidase and lactic dehydrogenase enzymes were monitored in the testis and kidney of adult albino rats. Results showed that aflatoxin B1 depressed the activity of alkaline phosphatase in both tissues, but increased that of acid phosphatase in only the testis. While GOT and 5'-nucleotidase were inhibited, GPT and lactic dehydrogenase activity was enhanced by this carcinogen. These responses were similar for the testis and kidney. The above findings coupled with the microscopical observation of the testis tissue seem to indicate that the essential lesion of this toxin on the testis may be a modification of the enzymes of germinal cells resulting from a gradual depletion of the latter. Furthermore, the results appear to show that by and large, aflatoxin B1 exerts only slightly different effects on the testis and kidney at the enzyme level.

Effect of 2,6-cis-diphenylhexamethylcyclotetrasiloxane on the reproductive organs of male mice.

Mice treated with tetrasiloxane, 20 and 40 mg/kg/day, showed lesions in the reproductive organs, cytological changes in the pituitary glands, and changes in levels of cholesterol, phosphomonoesterases, transaminases, 5'-nucleotidase, lactic and isocitric dehydrogenases, and ATPase.

The development and significance of transaminases and 5(1)-nucleotidase (5(1)-ribonucleotide phosphohydrolase) in chick testis.

The development of testicular glutamate oxaloacetate (GOT) and glutamate pyruvate (GPT) transaminases and of 5'-Nucleotidase was monitored in the male Nigerian fowl. The findings indicated triphasic and monophasic oscillations of GOT and GPT respectively with age. 5(1)-Nucleotidase was found to decrease with increase in the chronological age of the chicks. Furthermore, while GOT and GPT exhibited cubic and quadratic relationships respectively with age and testicular weights, 5(1)-Nucleotidase was related to both variables in a quadratic manner. The results are discussed in terms of spermatogenesis which may bear profoundly on the overall reproductive performance traits of the Nigerian cockerel.

Age-influence variations in the levels of cholesterol and ATPase activity in the testes of prepuberal chicks.

Changes in the levels of total, free and esterified cholesterol and ATPase activity were measured in male prepuberal Nigerian fowl. Results showed that in all cases, the testicular levels of these parameters increased to peak levels and decreased thereafter. Total and free cholesterol levels were maximal at 12 weeks while the esterification of cholesterol was maintained from 14 weeks through to the 16th week. The results of ATPase activity corroborated these findings. The data suggest that peak androgen formation occurred in these chicks between 14th--16th weeks of age. As evidenced by the higher percentages of esterified cholesterol at all the ages studied, it is suggested that the Nigerian male fowl is an early-maturing specie.

Age-dependent changes in the activities of ATPase and some pyridine nucleotide-linked enzymes in the chick testis.

The ontogeny of some of the enzymes connected with carbohydrate metabolism in the testis were studied in the White-Rock chicks. In the first place testicular growth in these chicks relate to their overall growth as measured by their body weights. ATPase and NAD+-dependent succinic dehydrogenase activities decreased both with advancing age and increasing testicular weight. However, these enzymes showed maximum activities at 17 and 28 weeks respectively. NAD+-linked isocitric dehydrogenase activity continually increased with increasing testicular weight and age. It is suggested that during spermatogenesis the activities of these enzymes are controlled by different developmental mechanisms.

The development of phosphomonesterases in the testes of prepuberal chicks.

1. The biochemical development and histochemical localisation of phosphomonoesterases in the testes of prepuberal chicks have been studied. 2. Maximum acid phosphatase activity was observed at 12 weeks with a decrease in enzyme activity after this age, whereas alkaline phosphatase activity fluctuated with age. 3. Acid phosphatase activity in chicks was similar to that of the cockerel in being tartarate-insensitive. 4. There was a low level of significant correlation between acid phosphatase activity and testes weight. 5. Both alkaline and acid phosphatase activities were observed in the basement membrane of the seminiferous tubules, and acid phosphatase activity also in the various spermatogenic elements. 6. The results suggest that acid phosphatase is more involved in spermatogenesis, and more widely distributed than alkaline phosphatase in testicular tissue during testicular development.