Neonatal exposure to Aroclor 1254: Effects on adult hepatic testosterone hydroxylase activities

1. The effects of neonatal exposure to Aroclor 1254 (100 μmol/kg) on the metabolism of testosterone by adult male and female rats were determined by comparing their hepatic microsomal testosterone hydroxylase activities at 60, 90 and 120 days after the initial exposure.

2. The most pronounced effects in male rats were observed 90 days after treatment with Aroclor 1254, whereas in female rats the major changes in testosterone hydroxylase activities were observed after 60 days.

3. Ninety-day-old male rats neonatally treated with Aroclor 1254 exhibited decreased basal testosterone 7α-hydroxylase and increased basal testosterone 16α-, 2α- and 15β-hydroxylase activities and androstenedione formation. In addition, the Aroclor 1254-mediated induction of testosterone 7α- and 6α-hydroxylase activities and androstenedione formation was decreased, and that of testosterone 2β- and 15β-hydroxylase activities was increased.

4. Sixty-day-old female rats exposed neonatally to Aroclor 1254 exhibited increased basal testosterone 16α-, 2β-, 6α- and 15β-hydroxylase activities and androstenedione formation, and increased Aroclor 1254-induced metabolism of testosterone at all positions except 16α and 2α.

5. Changes in testosterone hydroxylase activities indicative of permanent damage (or imprinting) in androgen metabolism, i.e. altered activities in 120-day-old animals, were observed only in male rats. These activities included basal testosterone 6β-, 16α- and 2α-hydroxylase activities and androstenedione formation.     

Studies on the role of acetylhydrazine in isoniazid hepatotoxicity.

Some factors affecting the metabolism of acetylhydrazine were studied in rats. This compound, a potent hepatotoxin, is a metabolite of isoniazid in man and is thought to be responsible for the hepatotoxicity of the drug. Isoniazid inhibited both the microsomal metabolism of acetylhydrazine, in vitro, and the acetylation to diacetylhydrazine in vivo. The overall effect of isoniazid in vivo was to increase metabolism of acetylhydrazine through the microsomal pathway leading to increased covalent binding of the toxic reactive intermediate to liver protein. The results suggest that the metabolism of acetylhydrazine produced as a metabolite of isoniazid may be quantitatively different from the metabolism of the compound alone. Preliminary studies in patients suffering isoniazid related liver damage indicated that acetylation of acetylhydrazine may be one of the factors involved in the hepatotoxicity of isoniazid in man.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced changes in the hydroxylation of biphenyl by rat liver microsomes

Biphenyl 2- and 4-hydroxylase activities and cytochrome P-450 concentrations in microsomes were increased by oral doses of less than 1 μg TCDD/kg. Female rats were more sensitive than male rats to the inductive effects of TCDD. since highly significant increases in biphenyl-hydroxylating activities were observed at the dose level of 0.2 μg TCDD/kg in female but not in male rats. The inductive effect was very persistent: biphenyl 2- and 4-hydroxylases remained stimulated even after 73 days following a single oral dose of 25 μg TCDD/kg. The levels to which the hydroxylases were stimulated in female rats were the same as in male rats. Rats of all ages from 10 to 335 days responded to hepatic microsomal effects of TCDD to approximately the same degree. The enzyme inductive effect was diminished by the simultaneous administration of actinomycin D. The K_m of biphenyl 2-hydroxylase (1.42 mM) was not altered significantly by TCDD treatment, but the K_m of biphenyl 4-hydroxylase (0.62 mM) was increased to approximately the same value (1.6 mM) as that of the 2-hydroxylase. The V_max of biphenyl 4-hydroxylase was increased 4.5-fold but that of biphenyl 2-hydroxylase was increased 16.5-fold. Rates of 2β- and 16α-hydroxylation of testosterone were suppressed by TCDD but rates of 7α- and 6β-hydroxylation were unaffected. It would appear that the hepatic microsomal mixed-function oxidases responsible for the hydroxylation of biphenyl and testosterone are different.     

Cholesterol 7α-hydroxylase—Evidence for a distinct hepatic microsomal enzyme system

The interrelationship of microsomal 7α-hydroxylation and drug oxidation reactions was studied in liver microsomes obtained from the male Wistar rat. When several compounds were administered to rats, a variety of changes ensued concerning the rate of cholesterol 7α-hydroxylation, ethylmorphine N-demethylase activity, and alterations in electron transport components. Cholestyramine and d-thyroxine administration resulted in significant increases in cholesterol 7α-hydroxylase activity. Both phenobarbital (PB) and spironolactone pretreatment did not produce an elevation of cholesterol 7α-hydroxylation, but significant increases in ethylmorphine N-demethylation over controls were realized. There was a lack of congruence with the rate of cholesterol 7α-hydroxylation and the content or activity of electron transport components whereas there was a demonstrable dependency of ethylmorphine N-demethylation on the monitored electron transport components for PB- and spironolactone-treated rats. Along with an elevation of cytochrome P448 content, 3-methylcholanthrene (3-MC) pretreatment reduced cholesterol 7α-hydroxylase activity and the rate of ethylmorphine N-demethylation. Concomitant treatment with 3-MC and cholestyramine caused no alteration of cholesterol 7α-hydroxylase activity. The inhibitory action of 3-MC on cholesterol 7α-hydroxylase activity was not due to competition for reducing equivalents in liver microsomes. The inhibitory phenomenon caused by NADH on microsomal cholesterol 7α-hydroxylase activity was reproducible and was dose-dependent at both subsaturating and saturating levels of NADPH. In conclusion, the results of this study indicate that the cholesterol 7α-hydroxylase enzyme system is distinctly different from that which catalyzes drug oxidations.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) effects on hepatic microsomal cytochrome P-448-mediated enzyme activities.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal mixed function oxidase (MFO) enzyme systems were examined in female rats. Although TCDD had little effect on NADPH-cytochrome c reductase activity and cytochrome P-450 content, the activities of the cytochrome P-448-mediated enzymes benzo[α]pyrene hydroxylase, ethoxyresorufin O-deethylase, and biphenyl 2-hydroxylase were greatly increased. Three months after a single oral dose of 2 μg/kg TCDD, the cytochrome P-450 content and benzo[α]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were still significantly increased. In addition, the microsomal metabolism of the novel substrate 4,4′-dimethylbiphenyl was greatly increased by TCDD pretreatment. Low dose studies revealed that the ED50 of TCDD induction of benzo[α]pyrene hydroxylase was 0.63 μg/kg and the lowest dose of TCDD which caused a significant increase in enzyme activity was 0.002 μg/kg. Studies in which [1,6-³H]TCDD was used to determine the extent of hepatic uptake of orally administered TCDD at the lowest effective dose of 0.002 μg/kg lead to the estimate that only 65 molecules of TCDD per hepatocyte were required to produce a measurable increase in benzo[α]pyrene hydroxylation. These results attest to the specificity and persistence of TCDD in the induction of cytochrome P-448-mediated enzyme activities in rat liver. The small number of molecules required to induce benzo[α]pyrene hydroxylase suggests that TCDD is among the most potent MFO-inducing agents yet demonstrated in mammalian liver.     

Influence of estradiol and testosterone on cytochrome P-450 and monooxygenase activity in immature brook trout,Salvelinus fontinalis

Levels of hepatic microsomal cytochrome P-450 were depressed by administration of estradiol-17β and were elevated by administration of testosterone in both male and female juvenile brook trout (Salvelinus fontinalis). Treatment-associated changes in the levels of other microsomal electron transfer components in liver did not reflect the changes in cytochrome P-450 content and were also distinct from the changes in these components in kidney. Electrophoretic analysis of hepatic microsomes revealed that estradiol treatment reduced the amounts of several proteins including some heme-staining protein at 56,000 daltons, possibly containing cytochrome P-450. Hepatic microsomal benzo[a]pyrene hydroxylase and the response to 7,8-benzoflavone in vitro were affected little by steroid treatment, and ethoxyresorufin O-deethylase activity could not be detected in any of the samples. Hepatic microsomes metabolized testosterone to a suite of products including 6β-hydroxytestosterone (the major metabolite) and 16β-hydroxytestosterone, plus as many as eleven unknown metabolites. Estradiol-17β treatment depressed the rates of testosterone metabolism and particularly the rates of 6β-hydroxylase activity but did not affect 16β-hydroxylase activity. Both activities were largely unaffected by testosterone. The results are consistent with the idea that both androgens and estrogens regulate the levels of hepatic cytochrome P-450 in brook trout and that the effect, at least of estradiol-17β, involves regulation of forms that function in specific hydroxylation of testosterone. The significance of these effects and whether factors additional to steroids are involved in this regulation of hepatic cytochromes P-450 in fish remain to be established.     

Differential action of progesterones on hepatic microsomal activities in the rat.

A significant reduction was found in the activity of drug-metabolizing enzymes (aminopyrine N-demethylase and coumarin 3-hydroxylase) and glucose 6-phosphatase in hepatic microsomes after the administration of reduced derivatives of progesterone (5α-pregnane-3β,-ol-20-one, 5β-pregnane-3α-ol-20-one, 5α-pregnane-3β,20β-diol and 5β-pregnane-3α,20α-diol) to rats. These steroids slightly raised inosine diphosphatase activity. On the other hand, 16α-hydroxyprogesterone and pregnenolone-16α-carbonitrile significantly increased drug metabolism and slightly elevated glucose 6-phosphatase. The contrasting action of the different progesterone derivatives was associated with changes in microsomal phospholipid synthesis. Pregnanolone and pregnanediol significantly decreased the de novo incorporation of [¹⁴C-Me]-l-methionine into microsomal phospholipids, mainly manifesting in phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine fractions; reduced the activity of S-adenosyl-l-methionine:microsomal-phosphatidylethanolamine methyl transferase; and caused a reduction of total microsomal phosphatidylcholine:phosphatidylethanolamine ratio. In contrast, 16α-hydroxy-progesterone and pregnenolone-16α-carbonitrile increased the de novo synthesis of microsomal phospholipids, methyl transferase activity and the ratio of total microsomal phosphatidylcholine: phosphatidylethanolamine. Treatment of rats with reduced progesterone derivatives diminished microsomal progesterone hydroxylation in the 16α- and 6β-position and raised progesterone Δ⁴-5α-dehydrogenase activity measured in vitro. On the other hand, 16α-hydroxyprogesterone and pregnenolone-16α-carbonitrile elevated progesterone hydroxylation. Considering these opposite effects it can be postulated that in the rat the induction of drug-metabolizing activity of the hepatic endoplasmic reticulum might be controlled by a balance displayed in the synthesis and metabolism of various progesterone derivatives.     

Effects of polybrominated biphenyls on metabolism of testosterone by rat hepatic microsomes

Polybrominated biphenyls (PBBs) have produced alterations in the male reproductive system of several species. These alterations may be a consequence of modified metabolism of androgens. The purpose of this investigation was to determine the effects of PBBs on hepatic microsomal metabolism of testosterone in vitro. Hepatic microsomes were prepared from male and female rats exposed to 0 or 100 ppm PBBs from Day 8 of gestation until they were killed at 1, 2, or 4 months of age. Testosterone and its metabolites were identified and quantified utilizing high-performance liquid chromatography or gas-liquid chromatography. Steroid identification was confirmed with gas chromatography/mass spectrometry. Hydroxylation of testosterone to 7α- and 6β-hydroxytestosterone in microsomes from both sexes at all ages was increased by pretreatment with PBBs. Microsomal conversion of testosterone to 16 α-hydroxytestosterone and androstenedione was enhanced by PBBs in females at all ages and in 1 month-old males but not from males of 2 and 4 months of age. Reduction of testosterone to dihydrotestosterone and dihydroandrosterone was inhibited in microsomes from both sexes at all ages by pretreatment with PBBs. These results support the contention that the effects of PBBs on the male reproductive system may be due, at least in part, to altered metabolism of testosterone.     

In vitro metabolism of benzo[a]pyrene and dibenzo[def,p]chrysene in rodent and human hepatic microsomes

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and often carcinogenic contaminants released into the environment during natural and anthropogenic combustion processes. Benzo[a]pyrene (B[a]P) is the prototypical carcinogenic PAH, and dibenzo[def,p]chrysene (DBC) is a less prevalent, but highly potent transplacental carcinogenic PAH. Both are metabolically activated by isoforms of the cytochrome P450 enzyme superfamily to form reactive carcinogenic and cytotoxic metabolites. Metabolism of B[a]P and DBC was studied in hepatic microsomes of male Sprague-Dawley rats, naïve and pregnant female B6129SF1/J mice, and female humans, corresponding to available pharmacokinetic data. Michaelis–Menten saturation kinetic parameters including maximum rates of metabolism (V_MAX, nmol/min/mg microsomal protein), affinity constants (K_M, μM), and rates of intrinsic clearance (CL_INT, ml/min/kg body weight) were calculated from substrate depletion data. CL_INT was also estimated from substrate depletion data using the alternative in vitro half-life method. V_MAX and CL_INT were higher for B[a]P than DBC, regardless of species. Clearance for both B[a]P and DBC was highest in naïve female mice and lowest in female humans. Clearance rates of B[a]P and DBC in male rat were more similar to female human than to female mice. Clearance of DBC in liver microsomes from pregnant mice was reduced compared to naïve mice, consistent with reduced active P450 protein levels and elevated tissue concentrations and residence times for DBC observed in previous in vivo pharmacokinetic studies. These findings suggest that rats are a more appropriate model organism for human PAH metabolism, and that pregnancy's effects on metabolism should be further explored.     

Differential effects of diabetes on microsomal metabolism of various substrates: Comparison of streptozotocin and spontaneously diabetic wistar rats

We have examined the effect of recent onset diabetes on several aspects of hepatic microsomal metabolism in both streptozotocin (STZ)-induced and spontaneously diabetic BioBreeding (BB) male and female Wistar rats. Differential alterations of the diabetic state on hepatic microsomal enzyme activities were observed. Female diabetic rats exhibited no change in benzo[a]pyrene (BP) hydroxylase activity, a decrease in testosterone Δ⁴-hydrogenase, and an increase in aniline hydroxylase. On the other hand, male diabetic rats demonstrated a decrease in hepatic BP hydroxylase activity, no change in testosterone Δ⁴-hydrogenase, and an increase in aniline hydroxylase. Insulin treatment corrected these effects. No change in kidney BP hydroxylase activity was apparent in either female or male diabetics. There were no marked differences between the chemically induced and genetic models of diabetes with respect to the metabolism studies. Serum testosterone levels were significantly lower than control in male BP diabetics, whereas no change was apparent in female diabetics. Light microscopy and serum insulin determinations indicated that the spontaneously diabetic animals we examined were not severely diabetic. From electrophoresis of hepatic microsomal proteins we determined that spontaneous diabetes of short duration does alter the protein distribution in the cytochrome P-450 region. We conclide that the acute effects of STZ-induced and spontaneous diabetes on hepatic microsomal metabolism are quantitatively and qualitatively similar, despite probable differences in etiology of the diabetic state.     

Metabolism of [14C]- and [36C]-labeled vinyl chloride in vivo and in vitro.

Label from [¹⁴C]vinyl chloride was covalently bound to protein and nucleic acids in vivo and in vitro in the presence of rat liver microsomal fractions or highly purified cytochrome P-450 and NADPH-cytochrome P-450 reductase preparations. The ratio of bound to total non-volatile metabolites increased in going from the in vivo to the microsomal to the purified system. [³⁶Cl]vinyl chloride was metabolized by microsomes and highly purified systems: no label was bound and most could be accounted for as chloride ion. Phenobarbital pretreatment of rats did not induce total metabolism of vinyl chloride in vivo at either 10 or 250 ppm exposure levels; however, binding to protein and RNA was enhanced at the 10 ppm but not the 250 ppm level. Phenobarbital pretreatment increased thein vitro microsomal conversion of vinyl chloride to both total and bound metabolites. A sizeable fraction of the label of [¹⁴C]vinyl chloride metabolized in vivo was recovered in the microsomal fraction of the liver, but sodium dodecyl sulfate polyacrylamide gel electrophoresis of in vitro incubations indicated that the metabolites were distributed among many microsomal proteins and not localized to cytochrome P-450. Evidence was obtained for the metabolism of the suspected vinyl chloride metabolite chloroethylene oxide by microsomal epoxide hydratase. However, the epoxide hydratase inhibitor 3,3,3-trichloropropylene oxide, which blocks the microsomal degradation of chloroethylene oxide, did not enhance the level of vinyl chloride bound to either protein or adenosine.     

Acetylhydrazine hepatotoxicity

Acetylhydrazine, a human metabolite of isoniazid, causes a dose-dependent centrilobular hepatic necrosis in phenobarbital-pretreated but not control rats. Ballooning degeneration and infiltration by inflammatory cells were confined to the centrilobular area. The portal area however was normal. In conjunction with this necrosis, acetylhydrazine also caused a dose-dependent decline in hepatic cytochrome P-450 in the phenobarbital-pretreated, but not control rats. Isoniazid (0.4 mmol/kg), given concomitantly, reduced the hepatotoxicity of acetylhydrazine. Rifampicin pretreatment did not increase the hepatotoxicity of acetylhydrazine but did cause a dose-dependent decline in hepatic cytochrome P-450.     

Tissue and sex differences in the activation of aromatic hydrocarbon hydroxylases in rats

Betamethasone and α-naphthoflavone produced similar activation of biphenyl 2-hydroxylase and benzo[a]pyrene 3-hydroxylase in control male rat liver microsomes. In small intestinal epithelial microsomes, betamethasone had no effect whereas α-naphthoflavone caused a pronounced activation of benzo[a]pyrene hydroxylation and a lesser activation of biphenyl 2-hydroxylation. In lung microsomes, betamethasone had no effect on either enzyme activity whereas α-naphthoflavone had no effect on biphenyl 2-hydroxylase but inhibited benzo[a]pyrene hydroxylase. In kidney cortex microsomes from male rats both compounds caused inhibition or had no effect whereas in kidney cortex microsomes from female rats betamethasone activated whereas α-naphthoflavone had no effect.Activation also occurred in isolated viable hepatocytes from male rats. The response of biphenyl 2-hydroxylase was very similar to that found in male rat liver microsomes but benzo[a]pyrene hydroxylase was more sensitive to activation and less sensitive to inhibition than in microsomes. The findings are interpreted as demonstrating the presence of more than one ‘latent’ aromatic hydrocarbon hydroxylase in rodents.     

Dose-dependent effects of medroxyprogesterone acetate on the hepatic drug-metabolizing enzyme system in rats

The activity of the hepatic microsomal drug metabolism was examined in vitro in rats pretreated with 10–600 mg/kg medroxyprogesterone acetate intraperitoneally daily for seven days. In both sexes there was a significant increase in the liver weight, amount of cytochrome P-450, activity of NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and 2,5-diphenyloxazole hydroxylase. The increase in 7-ethoxycoumarin-O-deethylase activity was also significant in female rats, but not in male rats. In the female rats pretreated with medroxyprogesterone acetate, the ability of α-naphtho-flavone and SKF 525A to inhibit benzo[a]pyrene hydroxylase was decreased and slightly increased, respectively. The results show that medroxyprogesterone acetate has a dose-dependent inducing effect on the hepatic drug metabolism in rats. Female rats seem to be more sensitive to the inducing effect of medroxyprogesterone acetate than the males. The characteristics of medroxyprogesterone acetate induction resemble mostly those caused by phenobarbital and pregnenolone-16α-carbonitrile.     

Enhancement of cholesterol turnover in rats by a catatoxic steroid (PCN) and a bile acid sequestrant (colestipol-HCl)

Catatoxic steroids induce hepatic microsomal enzymes. To determine if cholesterol catabolism to bile acids by microsomal enzymes is stimulated by a catatoxic steroid, effects of pregnenolone-16α-carbonitrile (PCN) alone or with colestipol-HCl on cholesterol 7α-hydroxylase, cholesterol synthesis and cholesterol turnover were studied. Male rats fed diets containing colestipol (1%), PCN (0.085%), colestipol plus PCN, or basic diet were injected with [1,2,-³H]-cholesterol complexed with serum lipoproteins. Serum cholesterol specific radioactivity was measured for 49 days. Hepatic cholesterol and bile acid synthesis were estimated by [1-¹⁴C]-acetate incorporation and cholesterol 7α-hydroxylase activity. Turnover curves were analyzed using a three-pool model. PCN significantly increased all rate constants and cholesterol production rate (10.75 to 12.87 mg/day), which in this model is a measure of total body cholesterol turnover. Colestipol significantly increased total body cholesterol turnover (10.75 to 19.91 mg/day) and the excretion rate constant (0.44 to 0.88 day^?1) and increased acetate incorporation 6-fold and cholesterol 7α-hydroxylase activity 2-fold. PCN only slightly inhibited the latter. Effects of colestipol plus PCN were not different from those of colestipol alone. No treatment significantly changed cholesterol serum levels overall. Colestipol results are consistent with reported data for bile acid sequestrants. PCN markedly increased cholesterol flux between pools; it does not appear to induce and may, in fact, inhibit bile acid synthesis, perhaps by decreasing availability of necessary cofactors or cholesterol substrate.     

Constitutive and induced hepatic microsomal cytochrome P-450 monooxygenase activities in male Fischer-344 and CD rats. A comparative study

The hepatic microsomal mixed function monooxygenase system (MFO) is the major enzyme system responsible for the activation and deactivation of xenobiotics. This study was designed to compare the hepatic MFO system in Fischer-344 and CD (Sprague-Dawley) rats. Hepatic microsomes were prepared from control, phenobarbital (3 × 80 mg/kg)-, and 3-methylcholanthrene (3 × 20 mg/kg)-pretreated male F-344 and CD rats (49 days old). Both control and phenobarbital-treated F-344 rats had significantly lower microsomal epoxide hydratase activity than corresponding preparations from CD rats. In addition, the pattern of benzo(a)pyrene metabolism was significantly different between the strains. Microsomes from F-344 rats produced less dihydrodiols and quinones than the corresponding preparations from CD rats. The spectral characteristics of cytochrome P-450 in hepatic microsomes from both control and phenobarbital-treated F-344 rats were significantly different from those observed in CD rat hepatic microsomes. Specifically, the λ_max for the reduced cytochrome P-450 CO complex occurred at a slightly longer wavelength and the reduced ETNC $\text{430}\text{455}$ nm peak ratios were larger by about 70%. No significant strain differences were detected in control rats or rats pretreated with either phenobarbital or 3-methylcholanthrene with regard to microsomal protein, benzphetamine-N-demethylase, NADPH-cytochrome c reductase, biphenyl-4-hydroxylase, biphenyl-2-hydroxylase, arylhydrocarbon hydroxylase, ethoxycoumarin- or ethoxyresorufin-O-deethylase activities. These results suggest that differences do exist in the in vitro hepatic microsomal metabolism of xenobiotics in these two strains of rats. These differences may be of importance with respect to the susceptibility of the two strains of rats to various toxic agents.     

Effects of inducers and inhibitors on the metabolism of aflatoxin B1 by rat and mouse.

The microsomal metabolism of aflatoxin B₁ (AFB₁) via various pathways and the induction and inhibition specificities of these pathways were examined in Sprague-Dawley rats, inbred strains of mice, and recombinant inbred lines derived from AKR/J and C57L/J cross. The data suggests that the metabolism of aflatoxin B₁ is catalyzed by at least three different enzymes of the microsomal mixed function oxygenase system: one that mediates conversion to aflatoxin M₁ (AFM₁-hydroxylase) is cytochrome P-448-linked and is associated with the Ah locus at the level of regulatory genetic factors: the other two enzymes which are cytochrome P-450-linked, mediate the metabolism of aflatoxin B₁ to aflatoxin Q₁ (AFQ₁-hydroxylase) and to AFB₁-2,3-oxide (metabolic activation). Although AFQ₁-hydroxylase and metabolic activation, measured in vitro as the formation of DNA-alkylating metabolite(s), are not clearly distinguishable on the basis of induction and inhibition responses, the two activities can be distinguished on the basis of their kinetic parameters and genetic regulation: (a) the apparent K_m of the metabolic activation pathway is almost three orders of magnitude (19.7 × 10^?4 M) higher than that of AFQ₁-hydroxylase (0.7 × 10^?4), [B. D. Roebuck and G. N. Wogan, Cancer Res.37, 1649 (1977)]; and (b) while both activities are induced by phenobarbital (PB) and depressed by 3-methylcholanthrene (3-MC), unlike the metabolic activation, differences in the 3-MC-induced depression of AFQ₁-hydroxylase activity were noted in Ah responsive and nonresponsive strains. Kinetic studies revealed that metabolic activation of AFB₁ is linear with the time of incubation up to 20 min, with up to 2 mg of microsomal protein in the incubation, and has a pH optimum of 7 to 7.4. While PB pretreatment, relative to control, enhanced the apparent V_max 3-fold, 3-MC depressed it about 60 per cent; however, the apparent K_m values of the three microsomal preparations were of the same order of magnitude (1.3 to 2 × 10^?3 M). Under the conditions of the incubation, the minimum number of binding sites in DNA was estimated to correspond to a resultant specific activity of 8–9 nmoles AFB₁-metabolite bound/μmole DNA-P.     

Role of diethyldithiocarbamate in ethylene dibromide metabolism and covalent binding

Diethyldithiocarbamate (DDC) pretreatment of rats prevented the ethylene dibromide (EDB) depression of liver glutathione (GSH) 2 hr after toxication. The levels of cytochrome P-450 seem to by unaffected by EDB. The steady-state kinetics of the reaction of GSH with [U-¹⁴C]EDB in vitro, in the presence of glutathione-S-transferase, was determined by GSH utilization and the formation of a labeled nonvolatile product. The inhibition of the enzyme by DDC is noncompetitive producing a change in V_max without a change in K_m. DDC also inhibited the covalent binding of [¹⁴C]EDB to microsomal proteins in the microsomal system supplemented with NADPH. EDB bound to microsomal protein in the absence of NADPH. The role of DDC in the metabolism and covalent binding of EDB to macromolecules is discussed.     

Studies on the nature of the in vitro enhancement of biphenyl 2-hydroxylation provoked by some chemical carcinogens

Studies on the metabolism of [¹⁴C]biphenyl and of 2-hydroxy and 4-hydroxybiphenyls confirm that preincubation of fresh hepatic microsomal preparations from rats or hamsters with chemical carcinogens such as safrole, benz[a]pyrene and 2-acetamidofluorene and a NADPH regenerating system produces an increase in the levels of 2-hydroxybiphenyl, through a specific increase in its formation from biphenyl. These data also support the validity of the fluorimetric assay method for monitoring this reaction. The addition of oestradiol or glutathione to the incubation mixture and the use of various preincubations and time periods with the carcinogens and NADPH prior to adding biphenyl indicate that production of an active metabolite of the carcinogens is probably a pre-requisite for the in vitro enhancement of biphenyl 2-hydroxylase to occur.The lack of effectiveness of EDTA in enhancing biphenyl 2-hydroxylase and the complete destruction of this enhancement by short-term storage of microsomal preparations at -20° suggests that the phenomenon is different from that of degranulation of the endoplasmic reticulum by carcinogens.     

Prostatic and hepatic metabolism of (1,2-3H) testosterone as affected by DDT pretreatment in the mouse

The 10-day oral administration of DDT (25 or 50 mg/kg) led to significant reductions in the accumulation of [1,2-³H]testosterone and its principal metabolite [1,2-³H]5α-dihydrotestosterone by the anterior prostate gland of the mouse. However, the ratio of [1,2-³H]testosterone to [1,2-³H]5α-dihydrotestosterone was not altered significantly. This would suggest that the uptake, but not the subsequent metabolism, of the labeled androgen was decreased. Hepatic formation of polar metabolites of [1,2-³H]testosterone was also reduced by DDT pretreatment. No significant changes were observed in accessory sex organ weights or prostate gland fructose concentration following pretreatment with this pesticide. The data suggest that DDT may in part alter the accumulation of prostatic androgens as a result of altered hepatic steroid hydroxylation. However, the results obtained do not rule out a direct effect of DDT on the uptake of [1,2-³H]-testosterone by the prostate gland.