Liver Microsomal Cytochrome P-450 Content Research Articles

Reversal of Experimental Ethanol‐induced Liver Steatosis by Borage Oil

The aim of study was to evaluate the hepatoprotective effect of borage oil containing predominantly gamma-linolenic acid in rats with alcoholic steatohepatitis. Liver of ethanol-treated animals was characterized by fatty and hydropic dystrophies. Liver triglyceride contents and activitiies of serum marker enzymes were significantly increased. Ethanol increased nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-induced chemiluminescence and the contents of liver thiobarbituric acid reactive substances (TBARS). The reduced glutathione content in the liver was decreased. Ethanol enhanced liver microsomal cytochrome P-450 (CYP450) content, aniline p-hydroxylase and amydopyrine-N-demethylase activities. The treatment with borage oil improved the liver morphology, decreased triglyceride contents and normalized serum marker enzyme activities. Borage oil developed an antioxidant effect in ethanol-treated rats. The treatment with this compound decreased NADPH-induced chemiluminescence and the content of lipid peroxidation products. Borage oil normalized CYP450 content compared with the ethanol-treated group. CYPI450 2E1 isoform is a main source of free oxygen radicals in the liver of ethanol-treated rats and we propose that the antioxidant effect of borage oil is realized via the normalization of CYP450 content and activities of CYP450-related microsomal oxidases, as borage oil can improve the lipid surrounding of CYP450. In our opinion, the hepatoprotection by borage oil in alcoholic steatosis is connected with its antioxidant properties.

Effects of atrazine on cytochrome P450 enzymes of zebrafish ( Danio rerio)

In this study, the effects of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in males and females of adult zebrafish ( Danio rerio) were studied. The liver microsomal cytochrome P450 content, NADPH-P450 reductase, aminopyrine N-demethylase (APND), and erythromycin N-demethylase (ERND) activity were measured. Zebrafish were exposed to control and 3 treatments (0.01, 0.1, and 1 mg L −1) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, within the range of test atrazine concentrations, either P450 content or P450 isozyme activities could be induced by atrazine. Compared to controls, P450 content was significantly increased at all atrazine concentrations at days 10, 15, and 20; thereafter, at day 25, all concentrations decreased to approximately the control levels, both in males and females. In addition, the strongest induction of P450 content was observed on day 15 in males and day 10 in females at treatment concentrations of 1 mg L −1. NADPH-P450 reductase activities showed mild increase in males; however, the females exhibited significant induction on days 15, 20, and 25; especially, at concentrations of 0.01 mg L −1, the induction level was consistently increased during the experiment. The inducements of APND and ERND in males were mainly observed on the days 5, 10, and 15, which showed less distinct induction, while significant induction was observed in cases of treatments during all days in females. In conclusion, atrazine induces P450 enzymes in zebrafish, and the effects may function as significant toxicity mechanisms in zebrafish. Additionally, it also confirms the importance of using a combined multi-time and multi-index diagnostic method to enhance the sensitivity and effectiveness of the indices adopted.

Dehydroepiandrosterone supplement increases malate dehydrogenase activity and decreases NADPH-dependent antioxidant enzyme activity in rat hepatocellular carcinogenesis

Beneficial effects of dehydroepiandrosterone (DHEA) supplement on age-associated chronic diseases such as cancer, cardiovascular disease, insulin resistance and diabetes, have been reported. However, its mechanism of action in hepatocellular carcinoma in vivo has not been investigated in detail. We have previously shown that during hepatocellular carcinogenesis, DHEA treatment decreases formation of preneoplastic glutathione S-transferase placental form-positive foci in the liver and has antioxidant effects. Here we aimed to determine the mechanism of actions of DHEA, in comparison to vitamin E, in a chemically-induced hepatocellular carcinoma model in rats. Sprague-Dawley rats were administered with control diet without a carcinogen, diets with 1.5% vitamin E, 0.5% DHEA and both of the compounds with a carcinogen for 6 weeks. The doses were previously reported to have anti-cancer effects in animals without known toxicities. With DHEA treatment, cytosolic malate dehydrogenase activities were significantly increased by ~5 fold and glucose 6-phosphate dehydrogenase activities were decreased by ~25% compared to carcinogen treated group. Activities of Se-glutathione peroxidase in the cytotol was decreased significantly with DHEA treatment, confirming its antioxidative effect. However, liver microsomal cytochrome P-450 content and NADPH-dependent cytochrome P-450 reductase activities were not altered with DHEA treatment. Vitamin E treatment decreased cytosolic Se-glutathione peroxidase activities in accordance with our previous reports. However, vitamin E did not alter glucose 6-phosphate dehydrogenase or malate dehydrogenase activities. Our results suggest that DHEA may have decreased tumor nodule formation and reduced lipid peroxidation as previously reported, possibly by increasing the production of NADPH, a reducing equivalent for NADPH-dependent antioxidant enzymes. DHEA treatment tended to reduce glucose 6-phosphate dehydrogenase activities, which may have resulted in limited supply for de novo synthesis of DNA via inhibiting the hexose monophophaste pathway. Although both DHEA and vitamin E effectively reduced preneoplastic foci in this model, they seemed to function in different mechanisms. In conclusion, DHEA may be used to reduce hepatocellular carcinoma growth by targeting NADPH synthesis, cell proliferation and anti-oxidant enzyme activities during tumor growth.

Profile of Territrem Metabolism and Cytochrome P-450 3A Expression in Liver Microsomes from Wistar Rats of Both Genders as a Function of Age

This study determined territrem metabolites after incubation of territrem A, B, or C with NADPH and liver microsomes from Wistar rat of both genders aged 2 to 76 wk. The liver microsomal cytochrome P-450 content, NADPH-cytochrome P-450 reductase activity, and CYP3A1 and CYP3A2 protein and mRNA levels were also analyzed. Male rats had significantly higher liver microsomal cytochrome P-450 content and NADPH-cytochrome P-450 reductase activities than females at 14 to 26 wk. Microsomal cytochrome P-450 content was decreased in senescence in both genders compared with postpubertal and adulthood stages. The activity of 6β-testosterone hydroxylase in male rats, which was significantly higher than those in females at all ages, decreased after 52 wk. After 26 wk, the levels of CYP3A1 protein markely declined in both genders, which resulted in a large gender difference (male greater than female). The protein levels and mRNA of CYP3A2 were constitutively expressed in 2- to 52-wk-old male rats, but they decreased after 76 wk, and decreased in females after 6 wk. The expression of CYP3A1 or CYP3A2 in males are generally higher than in females. The metabolites of territrems MA1, MAX, MA2, MB2, MB4, and MC were measured by high-performance chromatography (HPLC). Formation of MA1, MAX, and MA2 decreased after 52 wk in males, and MAX and MA2 were not formed after 6 wk in females. The amount of MB2 formed in females was less than in males, but the amount of MC (TRC metabolites) formed in females was higher than in males. The gender differences in metabolism of TRA were related to the protein and mRNA expression of CYP3A2. The protein levels and mRNA expression of CYP3A2 and efficiency of territrems metabolism were decreased after 76 wk. The results suggested that the effects of age and gender on territrem metabolism are due to differences in CYP3A1 and CYP3A2 expression in the liver microsomes. This work is supported by National Science Council (NSC 91-2314-B-109-101 and NSC 92-2314-B-109-101). The authors are indebted to Dr. Ling Kuo Huang and Dr. Thomas Barkas for reading the article and for critical suggestions.

Possible role of ginsenoside Rb1 on regulation of rat liver triglycerides.

We have studied the effects of ginsenoside Rb1 (GRb1) on the change in lipid contents in rat liver. When GRb1 was administered intraperitoneally to rats, liver microsomal cytochrome P-450 content and NADPH-cytochrome P-450 reductase activity were lower than those in control rats. The contents of triglyceride (TG) and cholesterol were decreased, but those of total phospholipid, phosphatidylcholine, and phosphatidylethanolamine were increased in the GRb1-treated group compared with controls. These results indicate that GRb1 might be involved in lipid metabolism by regulating the activity of microsomal cytochrome P-450 monooxygenase. Although liver TG levels were reduced by GRb1, the levels of TG and beta-lipoprotein in serum from the GRb1-treated group did not change as compared with those in controls. Thus we suggest that the decrease in liver TG levels with GRb1-treatment is not associated with the secretion of TG-rich very low-density lipoprotein. Furthermore, the level of cAMP was also significantly increased in the GRb1-treated group as compared with that in controls. Additionally, the cAMP level was more markedly increased as compared with that in the GRb1-treated group or control group when GRb, was exogenously added to the reaction system for measuring cAMP production in homogenates from control group liver. Accordingly, these results demonstrate that GRb1 might lower TG levels via cAMP-production in the liver, and GRb1 might be an interesting candidate to for a modulator of cAMP-mediated effects, especially within the liver steatosis system.

The effect of protoporphyrinogen oxidase inhibitors on microsomal and mitochondrial cytochromes.

Neurological dysfunction is a characteristic feature of acute porphyrias, unexplained until now. One of the possible explanations is a deficiency of heme in the central nervous system, caused by a block in porphyrin biosynthesis. To test this possibility, the content of brain mitochondrial cytochrome a3 was determined after intracerebroventricular administration of the protoporphyrinogen oxidase-inhibiting herbicide fomesafen. It was established in in vitro experiments that fomesafen is a potent inhibitor of brain mitochondrial protoporphyrinogen oxidase (PROTOOX). Addition of 10(-6) M fomesafen to incubation mixture decreased PROTOOX from 1.02 nmol/mg/h (controls) to 0.42 nmol/mg/h. 10(-5)M of fomesafen decreased this activity to 0.27 nmol/mg/h. In in vivo experiments, 5 microleters of fomesafen solution containing 0.2 M fomesafen/l was administered to male rats by intracerebroventricular injection. The content of brain mitochondrial cytochrome a3 was determined 72 hours later. A slight decrease of the a3 content was observed (control rats 0.25 nmol/mg protein, treated rats 0.22 nmol/mg). Brain cytochrome P450 activities were below detection limits in both control and treated groups. In a separate experiment, male ICR mice were fed 1000 ppm of the protoporphyrinogen oxidaseinhibiting herbicide oxadiazon in the diet for 10 days. Liver microsomal cytochrome P450 content was decreased and liver porphyrins increased. An increase of porphyrin content was also observed in the testes of oxadiazon-fed mice, but testicular cytochrome a3 content was unchanged. The results indicate that, contrary to liver microsomal cytochromes P450, the mitochondrial cytochromes are not susceptible to changes in heme biosynthesis.

Effect of Dietary Fat on Rat Liver Microsomal Enzymes and Lipid Composition.

Dietary fat is known to influence the activities of various membrane-bound enzymes. The impact of various commonly consumed vegetable fats of India, viz., groundnut, coconut, safflower or mustard oil with varied fatty acid composition on the activities of liver microsomal membrane-bound enzymes and microsomal lipid composition was studied. In a feeding experiment, male weanling rats were fed these oils at 20g per 100g of the diet for 4 months. Liver microsomal cytochrome P-450 content was found to be significantly higher in the safflower oil-fed group and lowest in the coconut- and mustard oil-fed groups. The activity of UDP-glucuronyltransferase was highest in the mustard oil- and safflower oil-fed groups and lowest in the coconut oil-fed group and intermediary in the groundnut oil-fed group. The activity of Mg2+-ATPase was markedly lower in the safflower oil-fed group than in the rest of the groups. Na+, K+-ATPase activity was distinctly higher in the mustard oil-fed group, while Mg2+-ATPase activity was markedly higher in the groundnut oil-fed group. Further, the liver microsomes of safflower oil and mustard oil-fed groups showed higher amounts of cholesterol and phospholipids than the other two groups. However, the safflower oil group showed a higher cholesterol to phospholipid molar ratio than the other groups. Changes in cholesterol to phospholipid molar ratio as well as in fatty acid composition of the microsomal membranes could be responsible for the differences in the activities of the enzymes observed in the different groups.

Effects of cytochrome P450 inducers on I-compounds in rat liver and kidney DNA.

I-compounds are covalent DNA modifications presumably derived from endogenous electrophiles. To investigate the possible role of cytochrome P450 in I-compound metabolism, groups of female Sprague-Dawley rats (225-250 g) were treated i.p. with vehicle or cytochrome P450 inducers, i.e. 80 mg/kg phenobarbital (PB), 20 mg/kg 3-methylcholanthrene (MC) or 50 mg/kg pregnenolone-16 alpha-carbonitrile (PCN), once daily for 4 days. DNA synthesis rate was measured via [3H]methylthymidine incorporation. DNA adducts and I-compounds in liver and kidney were analyzed 1 and 8 days after the last treatment. Total liver and kidney microsomal cytochrome P450 content and activities of representative drug-metabolizing enzymes for PB, MC and PCN, i.e. benzphetamine N-demethylase, ethoxycoumarin O-deethylase (ECD) and erythromycin N-demethylase, were also determined in all groups. PCN caused significant depletion of total non-polar I-compounds at 1 day, compared to controls. Levels of several individual I-spots in liver were differentially reduced by each of the three inducers at 1 day. Most I-spots were restored to control levels at 8 days. Kidney I-compounds were not affected by PB or PCN, but MC reduced the level of one non-polar individual I-compound at 1 day. Except for the expected DNA adduct formation from MC, there were no qualitative changes in profiles of postlabeled modified nucleotides. Total cytochrome P450 content in liver microsomes and activities of individual P450 enzymes were significantly increased by treatment with each of the inducers at 1 day. This was, however, not the case at 8 days in PB- and PCN-treated livers. MC-treated rats, on the other hand, displayed elevated levels of liver cytochrome P450 and ECD at 8 days. In kidney, PB and PCN did not elicit induction of P450 and individual enzymes, but MC increased total P450 content and ECD activity at 1 day, and ECD activity alone at 8 days. These results suggest a major role for cytochrome P450 enzymes in the metabolism of I-compounds.

Advances in pharmacological studies of silymarin

Silymarin is the flavonoids extracted from the seeds of Silybum marianum (L) Gearth as a mixture of three structural isomers: silybin, silydianin and silychristin, the former being the most active component. Silymarin protects liver cell membrane against hepatotoxic agents and improves liver function in experimental animals and humans. It is generally accepted that silymarin exerts a membrane-stabilizing action preventing or inhibiting membrane peroxidation. The experiments with soybean lipoxygenase showed that the three components of silymarin brought about a concentration-dependent non-competitive inhibition of the lipoxygenase. The experiments also showed an analogous interaction with animal lipoxygenase, thus showing that an inhibition of the peroxidation of the fatty acid in vivo was self-evident. Silybin almost completely suppressed the formation of PG at the highest concentration (0.3 mM) and proved to be an inhibitor of PG synthesis in vitro. In our experiments, silybin at lower dose (65 mg/kg) decreased liver lipoperoxide content and microsomal lipoperoxidation to 84.6% and 68.55% of those of the scalded control rats respectively, and prevented the decrease of liver microsomal cytochrome p-450 content and p-nitroanisole-O-demethylase activity 24 h post-scalding. Effects of silymarin on cardiovascular system have been studied in this university since 1980. P. O silymarin 800 mg/kg/d or silybin 600 mg/kg/d reduced plasma total cholesterol, LDL-C and VLDL-C. They however, enhanced HDL-C in hyperlipemic rats. Further studies showed that silymarin enhanced HDL-C but didn't affect HDL-C, a property of this component which is beneficial to treatment of atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Excessive Ascorbic Acid Dose on Liver Microsomal Mixed Function Oxidase System in Guinea Pigs

In the first experiment, guinea pigs were fed diets containing graded levels of ascorbic acid (AsA) from deficient to excess (0, 6, 60, 600, 6, 000, and 12, 000mg/kg of diet) for 15 days after pre-feeding with a low AsA diet (60mg/kg) for one week. Pentobarbital sleeping time measured on day 9 was shortened in the 600- to 12, 000-mg AsA groups as compared with the two AsA deficient (0 and 6mg) and 60-mg AsA groups. Liver microsomal cytochrome P-450 content increased with a dose-response relation to the dietary AsA level except in the excess AsA group (12, 000mg/kg). However, cytochrome b5 content and NADPH-cytochrome c reductase activity showed no significant response. Activities of aminopyrine N-demethylase and aniline hydroxylase showed the same response as seen in cytochrome P-450 content, but to a lesser extent. In the second experiment, guinea pigs were fed the diet containing the normal level of or excess AsA (300 or 24, 000mg/kg of diet, respectively) for 4, 9 or 15 days after a pre-feeding with the normal AsA diet for one week. No significant differences in the time courses of those responses described above were shown between the two AsA groups. In conclusion, the liver microsomal mixed function oxidase activity did not change even transiently during a short period after administration of a large excess of AsA such as 24, 000mg/kg of diet, and 300mg AsA/ kg of diet is enough to ensure normal levels of the liver microsomal mixed function oxidase system in guinea pigs.

Induction of xenobiotic-metabolizing enzymes and peroxisome proliferation in rat liver caused by dietary exposure to di(2-ethylhexyl)phosphate.

Exposure of rats to 1% or 3% (w/w) di(2-ethylhexyl)phosphate in the diet for five days results in two- to three-fold inductions of liver cytosolic epoxide hydrolase activity and microsomal cytochrome P-450 content. Cytochromes P-450b + e were induced 20- to 35-fold, but no increase was observed in cytochrome P-450c. Considerably smaller effects were obtained on NADPH-cytochrome c reductase, microsomal epoxide hydrolase and microsomal cytochrome b5 content, and there was no effect on cytosolic glutathione transferase activity, under the same conditions. A dramatic increase in cyanide-insensitive palmitoyl-CoA oxidation and total mitochondrial protein, together with smaller increases in total catalase and cytochrome oxidase activities, were observed after treatment with di(2-ethylhexyl)phosphate, indicating that this compound causes proliferation of both peroxisomes and mitochondria. It is suggested that the induction of cytosolic epoxide hydrolase and the proliferation of peroxisomes may be related processes.

In vitro studies on the metabolism and co valent binding of [ 14C]1,1-dichloroethylene by mouse liver, kidney and lung

The metabolism and covalent binding of 1,1-dichloro[1,2- 14C]ethylene (DCE) to subcellular fractions of liver, kidney and lung of C57BL/6N mice have been investigated in vitro. Covalent binding was NADPH- and cytochrome P-450-dependent. The microsomal fraction bound more radiolabel than any other subcellular fraction, and the levels of covalent binding in cell fractions correlated well with their cytochrome P-450 content. Covalent binding by mouse liver and lung microsomes also reflected their cytochrome P-450 content. However, although mouse kidney microsomes contained twice as much total cytochrome P-450 as the lung, no detectable covalent binding of DCE-derived radioactivity occurred in kidney. Omission of NADPH, heat inactivation of microsomes, carbon monoxide, addition of SKF-525A, piperonyl butoxide or reduced glutathione (GSH), all inhibited (40–90%) covalent binding of radiolabel to liver and lung microsomes. The absence of O 2 (incubation under N 2) did not greatly affect the metabolism and covalent binding. Pretreatment of mice with various inducers, phenobarbital (PB),β-naphthoflavone (β-NF), pregnenolone 16α-carbonitrile (PCN) and 3-methylcholanthrene (3-MC), evoked increases in total liver microsomal cytochrome P-450 content (2-fold) and corresponding increases in covalent binding (3-fold). However, microsomes from PCN-treated mice showed only a 50% increase in DCE binding. Kidney microsomes from control, PB-, and β-NF-pretreated mice were incapable of covalent binding of radiolabel but those from PCN- and 3-MC-pretreated mice showed levels of binding similar to untreated mouse lung microsomes. It is proposed that the nephrotoxicity of DCE may be due to translocation of reactive metabolites from the liver to the kidney.

Induction of rat hepatic drug metabolizing enzymes by substituted urea herbicides.

Effects of eight structurally closely related substituted urea herbicides were investigated on the induction of cytochrome P-450 dependent monooxygenase enzyme complex, as well as on two conjugating enzymes after short-term treatment of rats. Liver microsomal cytochrome P-450 content was induced approximately by 50%. Cytochrome P-450 dependent monooxygenase activities showed a great variety depending on the substrate and on the herbicide. Two-18-fold induction was detected with 7-ethoxycoumarin, while up to 8-fold induction was measured with benzo(a)pyrene. Aldrin epoxidase activities were increased up to 3-fold, and aminopyrine N-demethylase activities were only slightly different from the control level. UDP-glucuronyltransferase and glutathione S-transferase activities were enhanced up to 2-fold. The results indicate that chemical structure of the related substituted urea compounds, the number of halogen substituents on their phenyl group exert a strong influence on the induction of monooxygenases.

Increased activity of certain hepatic drug-metabolizing enzymes due to subchronic exposure to bromobenzene

Male Sprague-Dawley rats were treated i.p. with different doses of bromobenzene (0, 0.1, 0.2, 0.3 and 0.5 mmol kg ) in corn oil for 4 weeks. The animals were killed 24 h after the end of the treatment for hepatic drug metabolism studies. A significant induction of the activities of microsomal aniline hydroxylase (51%) and aminopyrine N-demethylase (58%) was observed at 0.5 mmol kg dose level. Liver microsomal cytochrome P-450 content was significantly increased (approx. 38%) at subchronic doses of 0.3 and 0.5 mmol kg . Hepatic glutathione (GSH) concentrations were increased (approx. 30%) in comparison to control values at subchronic doses of 0.3 and 0.5 mmol kg , whereas a significant induction (46–72%) of the activities of conjugating enzymes, cytosolic glutathione S-transferases was noticed at any subchronic dose level of bromobenzene studied. These results suggest that, under certain conditions, subchronic pretreatment with bromobenzene may have the potential to modify the acute toxicity of its own as well as those of other environmental pollutants which require metabolic activation.

Solubilization and partial purification of two forms of cytochrome P-450 from trout liver microsomes

1. 1. Liver microsomal cytochrome P-450 content, NADPH-cytochrome c reductase, aniline 4-hydroxylase and ethylmorphine N- demethylase activities of rainbow trout, Salmo gairdneri, were found to be 0.16 ( n = 10) nmol P-450/mg protein, 38 ( n = 5) units/mg protein, 0.04 ( n = 4) nmol p- aminophenol/min/mg protein and 0.174 ( n = 3) nmol formaldehyde/min/mg protein, respectively. 2. 2. Trout liver cytochrome P-450 was solubilized by treatment of microsomes with sodium cholate. Chromatography on DEAE-cellulose column yielded two distinct cytochrome P-450 fractions from solubilized microsomes. Cytochrome P-450-I was eluted with Emulgen 913-containing buffer. Application of 0.08 M KCl in Emulgen 913-containing buffer to the DEAE-cellulose column eluted cytochrome P-450-II fraction. Cytochrome P-450-I was further purified on hydroxylapatite column. 3. 3. CO-difference spectrum of dithionite-reduced cytochrome P-450-I gave a peak at 449 nm while the similar spectrum of cytochrome P-450-II showed a maximum absorbance at 451 nm. Monomer molecular weights of cytochrome P-450-I and cytochrome P-450-II were determined by sodium dodecyl sulfate gel electrophoresis and found to be 56,000 and 48,500, respectively.

Protective action of diethyldithiocarbamate and carbon disulfide against various nephrotoxic agents.

Diethyldithiocarbamate (DTC) and carbon disulfide (CS2) protected mice against nephrotoxicity induced by chloroform, 1,1-dichloroethylene (1,1-DCE), furan, bromobenzene but not cephaloridine. Nephrotoxicity of chloroform and 1,1-DCE, but not that of furan, was augmented in CCl4-poisoned mice, which was also protected by DTC and CS2. Bromobenzene nephrotoxicity was, however, suppressed in CCl4-poisoned mice. CCl4-poisoning produced a marked decrease in liver microsomal monooxygenase (MO) activities and cytochrome P-450 content, whereas some renal microsomal MO activities such as aniline hydroxylase and p-nitroanisole O-demethylase were rather increased. DTC and equimolar doses of CS2 reduced these renal microsomal MO activities and cytochrome P-450 content in both normal and CCl4-poisoned mice. Thus, nephrotoxicity of chloroform, 1,1-DCE and furan may result from metabolic activation of these nephrotoxins in the kidney MO system. Bromobenzene nephrotoxicity, however, may be evoked by active metabolites formed in the liver and transferred to the kidney. Renal microsomal bioactivation may not be required in the case of cephaloridine nephrotoxicity. The action of DTC, especially when given orally, may be mediated through CS2 produced in the stomach, which probably inhibits metabolic activation of some nephrotoxins in the kidney.

Toxicity and microsomal enzyme induction effects of several polybrominated biphenyls of firemaster

Some toxicological and pharmacological effects of 2,4,5,2′,5′-penta- (congener 1), 2,3,4,2′,4′,5′-hexa- (congener 5), 2,4,5,3′,4′,5′-hexa- (congener 6), 2,3,4,5,3′,4′,-hexa- (congener 7), and 2,3,4,5,2′,3′,4′-hepta-bromobiphenyl (congener 9) were evaluated in male rats given a single 90 mg/kg ip injection and killed seven days later. Only congener 7 depressed body weight gain, spleen and thymus weights, and caused severe histopathological changes in the thymus. Congener 7 caused the largest increase in liver weight and the most changes in liver pathology while congener 1 failed to enlarge this organ and caused the mildest ultrastructural changes. Liver microsomes were isolated and evaluated for enzyme induction from all treated rats except those administered congener 6, which was previously identified as a mixed-type enzyme inducer (Dannan et al., 1978b). All congeners increased the liver microsomal cytochrome P-450 content, but only congener 7 shifted the carbon monoxide difference spectrum absorption maximum to 448.0 nm. The microsomal ethyl isocyanide difference spectrum 455/430 nm ratio was increased the most by congener 7 (3 fold). All congeners increased cytochrome P-450 reductase and microsomal epoxide hydrase activities by nearly 1.5–3 fold. Congener 7 failed to induce aminopyrine-N-demethylase activity but the remaining congeners increased it by 2 fold. Congener 7 was the most effective inducer of benzo[a]pyrene hydroxylase and p-nitrophenol UDP-glucuronyl transferase. These results add to the suggestion that the presence of an ortho halogen on a polyhalogenated biphenyl does not completely abolish toxicity or 3-methylcholanthrene-type microsomal enzyme induction.

Phenobarbital induction of procyclidine metabolism: in vitro study.

After pretreatment of adult male Wistar rats with phenobarbital, a well-known cytochrome P-450 inductor, the liver microsomal cytochrome P-450 content increased significantly compared to that of control rats. At the same time the amount of procyclidine, metabolized by the 9000 g supernatant fraction of rat liver homogenate fortified with a NADPH generating system, increased significantly as well. However when related to the liver microsomal cytochrome P-450 content, the amount of metabolized procyclidine does not differ anymore between phenobarbital treated and control rats. Therefore phenobarbital induces the in vitro metabolism of procyclidine.

Liver microsomal enzyme induction and toxicity studies with 2,4,5,3′,4′-pentabromobiphenyl

A relatively minor component of Firemaster, 2,4,5,3′,4′-pentabromobiphenyl (PenBB), was purified and compared to the effects of Firemaster and 3,4,5,3′,4′,5′-hexabromobiphenyl (HBB). Male rats were given 90 mg/kg, ip, of PenBB or Firemaster and killed 2 weeks later. Blood samples were clinically analyzed and various tissues were examined for pathological effects. Induction of hepatic microsomal drug metabolizing enzymes and effects on the immune system were also investigated. No hematologic changes could be detected 2 weeks after either treatment. PenBB caused a significant loss in body weight, and the spleen weight to body weight and thymus weight to body weight ratios were slightly decreased by this treatment. Liver weight to body weight ratios were increased by both treatments. Ultrastructural changes in hepatocytes included proliferation of the smooth endoplasmic reticulum and the appearance of lipid droplets and concentric membrane whorls. The splenic T helper and B cells were substantially impaired by Firemaster and particularly by PenBB. There was a marked increase in liver microsomal cytochrome P-450 content and its CO difference spectrum absorption maximum was shifted to 448.5 nm. The microsomal ethyl isocyanide difference spectrum 455 430 -nm ratio was also increased. Benzo[ a]pyrene hydroxylase, p-nitrophenol UDP-glucuronyltransferase, and epoxide hydrolase activities were markedly increased. Less pronounced induction of NADPH-cytochrome P-450 reductase, and aminopyrine- N-demethylase activities occurred. These results, in addition to the results of SDS-polyacrylamide gel electrophoresis suggest that the mono- ortho-brominated congener is predominantly a 3-methylcholanthrene (MC)-like inducer and is toxic. In another experiment when enzyme induction by PenBB (30 mg/kg) was compared to that caused by 30 or 2 mg/kg of HBB it was evident that the non- ortho-substituted congener is the more potent MC-like inducer. Therefore, we conclude that the single ortho halogen of PenBB may weaken but not abolish its toxicity and MC-like enzyme induction effects.

The quantitation of liver cytochrome P450— LM2 mRNA in rabbits of different ages and after phenobarbital treatment

Using very young (neonate or 0 day), 50-, 100-, 300- and 830-day-old rabbits we have studied changes in liver drug-metabolizing activities. Total liver microsomal cytochrome P450 content, benzphetamine N-demethylase and 7-ethoxycoumarin 0-deethylase activities all decline with increasing age. When the rabbits of different age groups were pretreated with phenobarbital, liver microsomal enzyme activities increased in each group as compared with its control, but the total induced activities still decreased with increasing age. Using a specific assay for translatable cytochrome P450-LM 2 mRNA, we show that the age-dependent decrease in control and phenobarbital-induced enzyme activities is due to a decrease in the levels of translatable mRNA specific for cytochrome P450-LM 2. This is the first report on an age-dependent decrease in the level of a specific translatable mRNA. We do not know whether this decrease is due to decreased mRNA synthesis, or increased mRNA degradation.