Differential induction of cytochrome P-450 catalyzed activities by polychlorinated biphenyls and benzo [a]pyrene in B6C3F1 mouse liver and lung

The properties of some constitutive and inducible enzyme activities of liver and lung microsomes were determined in B6C3F1 mice pretreated by either intratracheal (i.t.) administration of benzo[a]pyrene (BaP) or polychlorinated biphenyl (PCBs) mixture (Aroclor 1254), or intraperitoneal (i.p.) administration with Aroclor 1254. After i.p. administration of Aroclor 1254, liver cytochrome P-450 content, aryl hydrocarbon hydroxylase (AHH), benzphetamine N-demethylase and nitroreductase activities were increased 2.8-, 2.0-, 2.2-, and 2.0-fold, respectively. Lung cytochrome P-450 content was also increased (1.9-fold) after i.p. administration of Aroclor 1254; AHH and nitroreductase activities, however, were not affected and benzphetamine N-demethylase activity was decreased. Aroclor 1254 administered i.t. did not affect liver cytochrome P-450 content. However, AHH and benzphetamine N-demethylase activities were decreased 1.4- and 1.2-fold, respectively, and nitroreductase activity was increased 1.6-fold. After i.t. administration of Aroclor 1254, lung cytochrome P-450 content and AHH activity were increased 1.4- and 2.2-fold, respectively. Benzphetamine N-demethylase activity was decreased 2.1-fold and nitroreductase activity was not affected. After i.t. administration of BaP, liver 7-ethoxyresorufin O-deethylase and nitroreductase activities were increased 2.2- and 1.5-fold, respectively, and benzphetamine N-demethylase activity was decreased 1.3-fold. Lung AHH and 7-ethoxyresorufin O-deethylase activities were increased 4.3- and 3.1-fold, respectively, and cytochrome P-450 content, benzphetamine N-demethylase and nitroreductase activities were decreased 1.4-, 1.2- and 1.3-fold, respectively, after BaP administration. These data indicate that different cytochrome P-450 isozymes induced in B6C3F1 mice are responsible for monooxygenase and nitroreductase activities, and that the route of administration of chemicals is important in the expression of cytochrome P-450 catalyzed activities.     

Effects of polychlorinated biphenyls on porphyrin synthesis and cytochrome P-450-dependent monooxygenases in small intestine and liver of Japanese quail

The effects of acute exposure to polychlorinated biphenyls (PCBs) on porphyrin synthesis and cytochrome P-450-dependent monooxygenases in the small intestine and liver were studied in male Japanese quail. The birds were dosed orally with the PCB mixture, Aroclor 1242, or the individual PCB isomers, 2,4,2',4'-tetrachlorobiphenyl (2-TCB) and 3,4,3',4'-tetrachlorobiphenyl (3-TCB), and were killed 48 h later. All the PCB compounds caused a significant increase in porphyrin content and delta-aminolevulinic acid synthetase (ALA-S) activity in the small intestine and liver. Increases in porphyrins were greater in the small intestine than in liver. However, a smaller increase in ALA-S activity occurred in the small intestine than in liver, suggesting that ALA-S induction is not a major mechanism for the increased porphyrin content of small intestine. All the test compounds significantly increased the cytochrome P-450 content of liver. In the small intestine, cytochrome P-450 content was increased by Aroclor 1242 and 2-TCB but not by 3-TCB. The activity of 7-ethoxyresorufin O-deethylase, however, was increased by all test compounds in both liver and small intestine. In contrast, there was a striking difference between small intestine and liver in the induction of 7-ethoxycoumarin O-deethylase (ECOD) activity by Aroclor 1242. In the liver, ECOD activity was unchanged or decreased, but in the small intestine, ECOD activity increased linearly with dose. No tissue difference in ECOD activity was observed after treatment with 2-TCB or 3-TCB. These findings suggest that acute exposure to a given PCB results in marked differences between small intestine and liver in porphyrin metabolism and in the induction of cytochrome P-450 isozymes and associated monooxygenases.     

Effect of host-mediating anti-tumor drugs on hepatic δ-aminolevulinic acid synthetase and heme oxygenase activity in mice

Effect of host-mediating anti-tumor drugs, OK-432 and PSK, on hepatic δ-aminolevulinic acid synthetase (ALA synthetase) and heme oxy genase activity was examined. Both OK-432 and PSK were shown to inhibit ALA synthetase activity when administered in vivo to mice. Both OK-432 and PSK also inhibited the 3,5-dicarboethoxy-1,4-dihydro collidine (DDC)-mediated induction of ALA synthetase when administered 12 and 16 h prior to the inducer. Additionally, the administration of OK-432 and PSK resulted in a significant increase in hepatic heme oxy genase activity.These data suggest that the decrease of hepatic drug-metabolizing enzymes as well as cytochrome P-450 levels seen on the administration of OK-432 and PSK is the result of the inhibition of ALA synthetase and of the increase of heme oxygenase activity leading to a decreased heme moiety required for the synthesis of cytochrome P-450 in the liver.     

Studies on the mechanisms of thallium-mediated inhibition of hepatic mixed function oxidase activity. Correlation with inhibition of NADPH-cytochrome c (P-450) reductase

Thallium (TlCl3) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome c (P-450) reductase and microsomal mixed function oxidase activities within 2-4 hr following treatment. These changes occurred independently of apparent effects on microsomal heme or cytochrome P-450 content, both of which remained unchanged with respect to control levels despite transient inhibition of delta-aminolevulinic acid (ALA) synthetase and induction of heme oxygenase. These results are consistent with the recognized properties of thallium as both a flavoprotein antagonist and sulfhydryl inhibitor and differ uniquely from the action of other metals which impair mixed function oxidase activity through compromise of heme biosynthesis and heme depletion. The potential utility of thallium compounds in further evaluating the functional characteristics of NADPH-cytochrome c (P-450) reductase and its role in microsomal oxidative processes is suggested from these observations.     

Effects of the chrysotherapeutic agents auranofin and gold sodium thiomalate on hepatic and renal drug metabolism and heme metabolism

These studies were designed to investigate the effects of the chrysotherapeutic agents auranofin and myochrysine (GST) on hepatic and renal drug-metabolizing enzymes and heme metabolism. Male Sprague-Dawley rats were either administered a single dose of auranofin (17, 34, or 68 mg/kg, p.o.) or administered daily doses of auranofin (0.2, 0.6, 2, 9, or 40 mg/kg/day, p.o.) or GST (1.2 or 5.8 mg/kg/day, i.p.) for 3 or 14 days. Rats were killed 24 h after the final treatment, and subcellular fractions of liver and kidney were prepared. Cytochrome P-450 (P-450) content and ethoxycoumarin-O-deethylase (ECOD), benzphetamine-N-demethylase (BPND), delta-aminolevulinic acid (ALA) synthetase, and heme oxygenase activities were determined. Twenty-four hours following single doses of auranofin, no effects on hepatic P-450, ECOD, or BPND were observed. Treatment with the positive control compounds, CoCl2 (60 mg/kg) and Co-protophorphyrin IX (33 mg/kg), produced decreases in all three variables at 24 hr. Auranofin, at 2 mg/kg, and GST treatment, at both doses, reduced hepatic P-450 and ECOD activity at 3 days. This effect was reversed with continued treatment for 14 days. BPND activity was unaffected at 3 days but was decreased at 14 days. Heme oxygenase activity was enhanced at 3 days and had returned to control activity at 14 days, while ALA synthetase was unaffected. With the exception of heme oxygenase, which was increased, renal variables were unaltered at 3 days. At 14 days, renal P-450 content was decreased in the high-dose auranofin group, heme oxygenase activity was increased in all groups, and ALA synthetase activity was elevated in high-dose auranofin animals. These data indicate that, at doses twenty times the human dose, auranofin and GST administration produced reversible decreases in hepatic and renal P-450 which may be the result of altered heme metabolism.     

Enhancement of the metabolism and hepatotoxicity of trichloroethylene and perchloroethylene.

Trichloroethylene anesthesia (1% for 2 hr) caused acute hepatic injury in rats pretreated with five different inducers of the hepatic mixed function oxidase system, phenobarbital, Aroclor 1254, hexachlorobenzene, 3-methylcholanthrene and pregnenolone-16-α-carbonitrile. Injury did not occur after trichloroethylene in rats pretreated with spironalactone or controls given vehicle alone. Morphologic liver injury was most severe in the phenobarbital- and Aroclor 1254-pretreated animals and was accompanied by marked perturbations in liver electrolyte content and more than 20-fold elevations in serum transaminase. Extent of serum transaminase elevation appears to relate directly to prolongation of anesthesia recovery time and the enhanced urinary excretion of trichlorinated metabolites. Metabolism of perchloroethylene (7.5 m-moles/kg, p.o.) was increased 5- and 7-fold, respectively, in phenobarbital- and Aroclor 1254-pretreated animals, but liver injury after perchloroethylene appeared only in Aroclor 1254 animals. Differential induction of various components of the microsomal mixed function oxidase system was quantified in parallel experiments using animals similarly pretreated with isomolar doses of the six inducers and the vehicle control and sacrificed at times corresponding to onset of chloroethylene exposure. Magnitude of induction of cytochrome P-450 among these seven groups of animals correlates with the mean extent of trichloroethylene-induced liver injury as quantitated by serum transaminases level (r = 0.95), with prolongation of anesthesia recovery time (r = 0.95Z) and with enhanced urinary excretion of trichlorinated metabolites (r = 0.88).     

Regulation of heme metabolism and cytochrome P-450 levels in primary culture of rat hepatocytes in a defined medium

Liver cells were prepared from adult Sprague-Dawley rats and used for the determination of delta-aminolevulinic acid synthetase (ALAS) activity and cytochrome P-450 concentrations at different time intervals in tissue culture in a serum-free synthetic medium. During the first 24 hr in culture, the level of cytochrome P-450 decreased to 30-40% of the level in isolated liver cells from untreated animals. The disappearance of cytochrome P-450 was especially fast in hepatocytes obtained from female phenobarbital-treated rats where only 40% of the original cytochrome P-450 was present after 2 hr in culture and 80% had disappeared in 2 days. The activity of ALAS increased 3- to 4-fold when measured 2 hr after plating, and it reached the maximum level in 19-24 hr when its activity was about eight times the original activity. In 2-4 days in culture, the activity of ALAS was four to five times above the original level. When the amount of delta-aminolevulinic acid (ALA) in the medium was increased from 1 to 100 microM, a decrease in ALAS was obtained, but no significant increase in cytochrome P-450 level was observed. Addition of heme to the medium gave a dose-dependent decrease in the activity of ALAS. Our data indicate that during the first 24 hr in culture the increase of ALAS activity was prevented by exogenous heme. This effect may be due to inhibition of the catalytic activity, suppression of the synthesis of the enzyme, or accelerated breakdown of the enzyme by heme.     

Alterations in hepatic heme and cytochrome P-450 metabolism in murphy-sturm lymphosarcoma-bearing rats implications for drug metabolism

Previous studies have shown that tumor-bearing rats have significantly decreased hepatic microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity with, consequently, significantly decreased capacity for microsomal oxidative drug metabolism. Subsequent investigations have revealed that the rates of hepatic cytochrome P-450 apo-protein synthesis and degradation are decreased significantly and hepatic microsomal heme oxygenase activity is increased significantly in rats bearing an extra-hepatic tumor. Further studies have been done to attempt to clarify the pathogenesis and significance of these observations. Hepatic delta-aminolevulinic acid (ALA) synthetase activity in male Wistar rats declined to a nadir of 162 ± 34 (S.E.) pmoles ALA per mg protein per 30 min 6 days following i.m. transplantation of Murphy-Sturm lymphosarcoma (vs control = 218 ± 36 pmoles per mg per 30 min). Turnover of ³H-labeled heme in microsomal CO-binding particles (i.e. cytochrome P-450 heme) was increased significantly 8 days following i.m. transplantation of Murphy-Sturm lymphosarcoma with a T_{$\text{1}\text{2}$} of 5.5 hr for the fast phase of hepatic cytochrome P-450 heme disappearance in tumor-bearing rats as compared with a T_{$\text{1}\text{2}$} of 7 hr in control rats. Hepatic cytochrome P-450 apo-protein concentration was slightly, but not significantly, increased in Murphy-Sturm lymphosarcoma-bearing rats as compared with control rats up to 10 days following tumor transplantation. These results suggest that, in Murphy-Sturm lymphosarcoma-bearing rats, decreased microsomal cytochrome P-450 concentration is the result of both decreased cytochrome P-450 apo-protein synthesis and increased cytochrome P-450 heme turnover. Apo-cytochrome P-450 concentration was not appreciably altered because increased cytochrome P-450 heme turnover and decreased cytochrome P-450 apo-protein degradation were balanced by decreased cytochrome P-450 apo-protein synthesis. Because of their effects on cytochrome P-450 concentration and action, these alterations in heme and hemoprotein metabolism may be of importance in regulating oxidative drug metabolism in the tumor-bearing state.     

Cytochromes P-4501A, P-4503A and P-4502B in liver and heart of Mugil capito treated with CYP1A inducers

Hepatic microsomes of Aroclor 1254-treated Mugil capito showed a single protein band detected in immunoblot with monoclonal antibody 1-12-3 to teleost (scup) CYP1A. The hepatic CYP1A like protein was induced with dose dependency after exposure of the fish to β-naphthoflavone (BNF) as well as to Aroclor 1254. The induced mullet hepatic CYP1A protein was confined to a distinct fraction obtained by DE-52 anion exchange chromatography, and its relative content in that fraction increased in fish that were treated with higher doses of inducer. EROD (7-ethoxyresorufin O-deethylase) activity in hepatic microsomes from mullet treated with various doses of BNF correlated significantly (r(2)=0.81502, P<0.01) with CYP1A content. Treatment of the mullet with low dose of Aroclor 1254 (25 mg/kg) induced only traces of CYP1A in liver microsomes (5.1±4.8 mg/kg). However, in mullet treated with the high dose of Aroclor 1254 (100 mg/kg) there was a dramatic induction in CYP1A content (408±275 pmol/mg) and this hemoprotein comprised about 83% of the total P-450 content of liver microsomes. The total level of P-450, although induced in the liver tissue, was not induced in heart tissue of Aroclor 1254 treated mullet. On the other hand, P-4501A was induced in treated mullet to a level that comprised almost all of the cardiac P-450 content. EROD activity in the heart tissue of induced mullet was characterized by low V(max) and high K(m) values (K(m)=2.35 mM, V(max)=39.5 pmol/min per mg) compared to the values recorded for the enzyme from the liver (K(m)=1.0 mM, V(max)=288.0 pmol/min per mg). Cardiac CYP1A with low catalytic activity and repression of CYP-types other then CYP1A in heart of CYP1A induced fish may be part of a mechanism aimed to preserve crucial levels of electron donors and molecular oxygen in cardiac muscle of fish exposed to CYP1A inducers.     

Selective induction and inhibition of liver and lung cytochrome P-450-dependent monooxygenases by the PCBs mixture, Aroclor 1016

The effects of the widely used industrial PCBs mixture, Aroclor 1016, as modifiers of monooxygenases were studied in rats and rabbits. From data presented, it is not possible to generalize the biological effects of PCBs observed with rats, namely, that they are potent, non-specific inducers of monooxygenase activities. In rat liver, Aroclor 1016 exhibited primarily the potent inducing effects of the barbiturate class of inducers. In contrast, in rabbits pretreated with Aroclor 1016, although cytochrome P-450 content of the liver was significantly increased, benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities were decreased 30-35%; and no changes in the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin were observed. These results strongly suggest differences in the regulation of cytochrome P-450 isozymes in the liver by the PCBs. A similar conclusion can be drawn from the pulmonary studies of Aroclor 1016-pretreated rabbits. In the lung, cytochrome P-450, form 2 and associated enzymic activities were markedly decreased, with little or no effect on the form 5 isozyme. Electrophoretic and chromatographic studies confirmed these findings. The induction and the repression of a form or forms of cytochrome P-450 by environmentally-derived chemicals may be important determinants of organ-targeted chemical toxicity.     

Contribution of methylsulfonyl metabolites of m-dichlorobenzene to the heme metabolic enzyme induction by the parent compound in rat liver

In the present study, we investigated the contribution of methylsulfonyl metabolites derived from m-dichlorobenzene (m-DCB) on the heme metabolic enzyme induction by the parent compound in rats. The time courses of the effects of a single ip administration of m-DCB (200 mg/kg, 1.36 mmol/kg) and 2,4- and 3,5-dichlorophenyl methyl sulfones (2,4- and 3,5-DCPSO2Mes) (each 50 mumol/kg) on hepatic microsomal cytochrome P450 content were almost in parallel with those on the total heme content in liver microsomes. m-DCB significantly increased the heme oxygenase activity, but 2,4- and 3,5-DCPSO2Mes did not. On the other hand, m-DCB and both methyl sulfones markedly enhanced the delta-aminolevulinic acid (ALA) synthetase activity. No change was observed in percentage saturation of the tryptophan pyrrolase activity after administration of m-DCB, whereas this ratio at 6 hr after injection of 3,5-DCPSO2Me was increased. In the liver of the DL-buthionine-(S,R)-sulfoximine (BSO)-treated rats dosed with m-DCB, both of 2,4- and 3,5-DCPSO2Mes were present at significantly lower concentrations than in non-BSO-treated rats. Additionally, the m-DCB did not elevate the ALA synthetase activity in the BSO-treated rat. On the other hand, the administration of either 2,4- or 3,5-DCPSO2Mes to BSO-treated rats resulted in induction of ALA synthetase. m-DCB and 2,4- and 3,5-DCPSO2Mes produced a dose-related increase in liver levels of methyl sulfones. The changes in the ALA synthetase activity after the administration of varying doses of m-DCB were similar to those after the administration of 2,4- or 3,5-DCPSO2Mes, whereas the sum of the concentration of two methyl sulfones in the liver of rats dosed with m-DCB was almost the same as the concentration of methyl sulfone after the administration of either 2,4- or 3,5-DCPSO2Mes. The results strongly suggest that the methyl sulfones derived from m-DCB, i.e., 2,4- and 3,5-DCPSO2Mes, contribute highly to the induction of the ALA synthetase activity by the parent compound.     

Acute effect of amitriptyline, phenobarbital or cobaltous chloride on delta-aminolevulinic acid synthetase, heme oxygenase and microsomal heme content and drug metabolism in rat liver

Cobaltous chloride (CoCl2) caused very marked decreases of cytochrome P-450, b5 and total heme contents and an increase of heme oxygenase activity. On the contrary, phenobarbital (PB) increased hepatic drug-metabolizing enzymes, but the total heme content remained unchanged. On the other hand, amitriptyline (AMT) caused a marked increase of delta-aminolevulinic acid (delta-ALA) synthetase activity at 12 and 24 hr. In addition, the contents of total heme and cytochrome b5 and the activities of aminopyrine (AM) N-demethylase and aniline (AN) hydroxylase at 24 hr were also increased by AMT, whereas cytochrome P-450 content did not change. This may be explained by the fact that AMT would increase hepatic heme synthesis through the prolonged induction of delta-ALA synthetase, but it may not cause an increase in cytochrome P-450 heme because there are increases in the contents of cytochrome b5 and total heme.     

Inhibition of rat testicular heme synthesis and depression of microsomal cytochrome P-450 by estradiol benzoate

Twenty-four hours after a single dose (50 μg, s.c.) of estradiol benzoate (EB), rat testicular microsomal heme and cytochrome P-450 were decreased to 72 and 76% of control levels respectively. Treatment of rats with human chorionic gonadotropin (hCG) resulted in elevated levels of microsomal heme and cytochrome P-450 and increased activity of δ-aminolevulinic acid (ALA) synthase (EC 2.3.1.37). However, the hCG-mediated elevations of testicular microsomal heme and cytochrome P-450 content failed to occur in animals treated with EB. To investigate the possibility that the observed effect of EB was mediated through the pituitary, studies were conducted with hypophysectomized animals. The increased microsomal heme and cytochrome P-450 content mediated by hCG in hypophysectomized animals was again prevented by administration of EB. The elevated activity of testicular mitochondrial ALA synthase produced by hCG in both intact and hypophysectomized animals was not affected by EB. Incorporation of [¹³C]ALA into microsomal heme was depressed 60% 12 hr following a single dose of EB (50 μg, s.c.). These data suggest that EB depresses testicular microsomal heme and cytochrome P-450 content by inhibiting the synthesis of heme at an enzymatic reaction other than ALA synthase.     

Effects of 1,2-dibromo-3-chloropropane on hepatic heme synthesis

Previous studies showed that 1,2-dibromo-3-chloropropane (DBCP) caused a decrease in hepatic microsomal cytochrome P-450 [D.E. Moody, B. Head, and E.A. Smuckler (1979) J. Environ. Pathol. Toxicol. 3, 177-190; D.E. Moody, G.A. Clawson, C.H. Woo, and E.A. Smuckler (1982) Toxicol. Appl. Pharmacol. 66, 278-279], suggesting that hepatic heme metabolism may be affected by DBCP treatment. This study tested this hypothesis. Various parameters of hepatic heme synthesis were measured at intervals ranging from 0 to 72 hr in male Sprague-Dawley rats given a single oral dose (200 mg/kg) of DBCP. Incorporation of the radiolabeled heme precursor [delta-14C]aminolevulinic acid (14C-ALA) into liver, protein, extracted heme, and subcellular fractions of liver homogenates was significantly decreased to 75, 58, and 81% of controls, respectively, at 24 hr. At 48 and 72 hr after DBCP treatment, the accumulation of 14C-ALA label after 4 hr in liver homogenates and subcellular fractions was significantly increased in comparison to controls. These changes in 14C-ALA uptake were accompanied by decreases in total liver and microsomal heme, but not mitochondrial heme. Decreases were found in the spectral content of two heme proteins, cytochromes P-450 and b5, and the activity of another heme protein, catalase. Heme oxygenase activity increased to 130, 151, 209, and 186% of control values at 12, 24, 48, and 72 hr after DBCP, respectively. A slight, but significant, increase in ALA-synthetase to 112% of controls occurred at 24 hr, and slight, but significant, decreases in ALA-dehydratase to 90 and 80% of control occurred at 12 and 24 hr, respectively. No significant changes in uroporphyrinogen-1-synthetase or ferrochelatase at the time points tested was noted. The porphyrin content of liver was increased to 130% of control, while the serum and urine porphyrin levels were decreased to 30% of the control values at 24 hr. Liver ALA content was not significantly altered through the time period studied, but serum and urine levels were increased at 24 hr to 176 and 130% of the control values, respectively. In conclusion, the decreases in liver heme proteins following a single oral dose of DBCP are accompanied by alterations in heme turnover, particularly a prolonged increase in heme oxygenase activity.     

Mechanisms of diethyldithiocarbamate-induced loss of cytochrome P-450 from rat liver

A single, intraperitoneal injection of diethyldithiocarbamate (DDTC) to adult, male Sprague-Dawley rats decreased hepatic cytochrome P-450 (P-450) concentrations. This effect was dose-dependent over a range of 250 to 750 mg/kg and most prominent 24-36 hr after dosing. Depletion of hepatic glutathione (GSH) by diethylmaleate (DEM) administration significantly decreased P-450 8 hr after concurrent treatment with DDTC at a dose which given alone had little effect on P-450 concentrations. When hepatic microsomes were incubated with DDTC in the presence of NADPH, P-450 was converted to cytochrome P-420 (P-420). Similar incubations employing [35S]DDTC demonstrated strict NADPH-dependent binding of labeled sulfur to microsomal membranes, suggesting that diminished P-450 concentrations are related to the metabolic activation of DDTC. Addition of reduced GSH to incubation mixtures blocked the binding of 35S to microsomal membranes, as well as conversion of P-450 to P-420. DDTC inhibited NADPH-ADP3+ mediated peroxidation of microsomal lipids in vitro, suggesting that the effect of DDTC on P-450 does not result from stimulation of lipid peroxidation, but may be influenced by the levels of hepatic GSH. DDTC treatment 1 hr after P-450 was pulse labeled by an intravenous injection of [3H]delta-aminolevulinic acid resulted in a 2-fold increase in the rate of loss of radioactivity associated with membrane-bound P-450 heme during the next 20 hr. Within this time interval, hepatic heme oxygenase (HO) activity increased and at 8 hr after dosing was 7-fold greater than control values in the livers, but was unchanged in the kidneys and spleens of DDTC-treated animals. An elevation of hepatic delta-aminolevulinic acid synthetase (delta-ALAS) activity occurred at 8 and 24 hr after DDTC treatment. Since this enzyme is rate limiting in the biosynthesis of heme, its increased activity may represent a compensatory response to offset the DDTC-mediated loss of P-450 heme.     

Persistent effects of a single dose of Aroclor 1254 on cytochromes P450IA1 and IIB1 in mouse lung.

The polychlorinated biphenyl mixture Aroclor 1254 has been shown to elicit prolonged biochemical responses in several rodent species, particularly induction of mixed function oxygenases in hepatic tissue. Lung is also of interest since a single dose of Aroclor 1254 has been demonstrated to have a tumor promoting effect, increasing the numbers of lung tumors in Swiss mice initiated with N-nitrosodimethylamine. To investigate the enzyme induction response in lung, male Swiss mice were given a single 100 or 500 mg/kg dose of Aroclor 1254 and euthanized at time intervals ranging from 48 hr to 30 weeks. Both cytochromes P450IA1 and IIB1 were followed by use of specific enzyme activities and Western immunoblotting. The IA1 isoform, as quantified by ethoxyresorufin-O-deethylase activity and immunoblotting with monoclonal antibody 1-7-1, was significantly elevated for 30 weeks after both doses. In contrast, benzyloxy-resorufin-O-dealkylase activity (P450IIB1 specific), which is constitutively expressed in rodent lung, was unaffected by Aroclor treatment at the lower dose at early time points, but induced twofold at 30 weeks. At the higher dose, however, enzymatic activity was decreased to 50% of control values, an effect which persisted for 4 weeks postexposure. These changes were confirmed by Western immunoblotting utilizing monoclonal antibody 2-66-3. Concomitantly, content of individual PCB congeners in lungs and carcass was quantified by gas chromatography with electron capture detection. One congener, 2,3,3',4,4'-pentachlorobiphenyl, was selectively retained in lung compared to carcass. Lack of correlation between changes in lung content of PCBs and levels of the P450 isoforms suggested interactions between congeners in control of P450 induction and repression. These data confirm a prolonged P450 induction response in nonhepatic tissue following Aroclor exposure, and further suggest a bidirectional role for certain PCB congeners in the regulation of P450IA1 and P450IIB1 expression in lung tissue.     

Sex-related effect of cadmium on hepatic cytochrome p-450, drug-metabolizing enzymes and 5-aminolevulinic acid synthetase and heme oxygenase in the rat

The effect of cadmium on hepatic cytochrome P-450 containing drugmetabolizing enzymes, δ-aminolevulinic acid synthetase (ALA synthetase) and heme oxygenase activity in both male and female rats was examined to define the nature of sex differences in response. In male rats injected i.p. with a single dose of cadmium chloride (1.25 mg Cd/kg body weight) there was a reciprocal relationship between the decrease of cytochrome P-450 levels and drug-metabolizing enzymes and the increase of heme oxygenase activity for up to 120 h. Hepatic ALA synthetase activity was initially decreased and returned to control levels at 24 h. In female rats, cadmium initially produced similar effects to those seen in male rats, but these were of shorter duration except for the increase of heme oxygenase activity. In addition, ALA synthetase activity was slightly increased at 24 h and 48 h.The results suggest that the sex differences in response reside mainly in the duration of the metal effect on these hepatic enzymes.     

Elevation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) polychlorinated biphenyls. Structure-activity relationships

Administration of the commercial polychlorinated biphenyl (PCB) Aroclor 1254 to immature male Wistar rats resulted in increased levels (80-110%) of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) hepatic cytosolic receptor protein which remained elevated for 14 days. The effects of structure on the activity of individual PCB congeners to modulate hepatic cytosolic receptor levels were compared to the structure-activity relationships (SARs) which have been developed previously for PCBs as inducers of hepatic microsomal monooxygenases. 3,3',4,4'-Tetra- and 3,3',4,4',5-pentachlorobiphenyl induced the cytochrome P-448-dependent monooxygenase, ethoxyresorufin O-deethylase (EROD), and resembled 3-methylcholanthrene in their mode of monooxygenase enzyme induction. These congeners also bound to the receptor protein; however, neither compound increased hepatic cytosolic receptor protein levels. Several PCB congeners which exhibit low binding affinities for the cytosolic receptor protein resembled phenobarbitone (PB) in their mode of monooxygenase enzyme induction and, like PB, elevated cytosolic receptor protein levels. Nevertheless, a comparison of the time course of monooxygenase enzyme induction and receptor protein elevation by 2,2',4,4',5,5'-hexachlorobiphenyl and PB illustrated significant differences in their activities. PB-mediated elevation of receptor levels was maximized 24 hr after the last dose, and 48 hr later the receptor levels decreased to control values. In contrast, 5 days after administration of a single dose of 2,2',4,4',5,5'-hexachlorobiphenyl (300 mumoles/kg) the receptor levels were elevated significantly, and these increased levels (205-127% increases over control) persisted for 14 days. There was no correlation between increased levels of hepatic receptor protein and the induction of the cytochrome P-450-dependent monooxygenases, aldrin epoxidase or 4-dimethylaminoantipyrine N-demethylase. Two PCBs, 2,3,3',4,4',5- and 2,2',3,4,4',5-hexachlorobiphenyl, which resembled Aroclor 1254 in their mode of monooxygenase enzyme induction, also elevated hepatic receptor protein levels but were less active than the PB-type inducers. Thus, the SARs developed for PCBs which elevate cytosolic receptor levels demonstrate that the most active compounds exhibit the lowest affinity for the receptor protein and do not induce EROD. In contrast, the more toxic PCB congeners which are approximate isostereomers of 2,3,7,8-TCDD both induced EROD and bound with high affinity to the receptor protein but did not increase hepatic cytosolic receptor protein levels.     

2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated depression of rat testicular heme synthesis and microsomal cytochrome P-450

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces hirsutism, alopecia, and chlorance, symptoms that suggest a possible alteration of endocrine function. Therefore, the effects of TCDD on rat testicular cytochrome P-450 content were investigated. Forty-eight hours after a single, oral dose of TCDD (25 μg/kg) testicular microsomal cytochrome P-450 levels were depressed by approximately 24%. Microsomal cytochrome P-450 continued to decrease to 62% of control levels at 4 days and remained at approximately the same levels 7 days following treatment. Testicular microsomal heme content exhibited a similar pattern after administration of TCDD. No alterations in testicular δ-aminolevulinic acid (ALA) synthase were detected. The incorporation of [¹⁴C]ALA into microsomal heme was decreased to approximately 36% of control values at 24 hr after TCDD administration. Testicular weights were not altered during the 7-day experimental period. These data suggest that TCDD depresses cytochrome P-450 levels in the rat testis through an inhibition of the synthesis of testicular heme.     

Hepatic microsomal metabolism and covalent binding of 2,4-dinitrotoluene

The effects of 2,4-dinitrotoluene (2,4-DNT) on xenobiotic metabolizing enzymes and the hepatic metabolism and covalent binding of this compound to microsomal proteins in vitro were studied. Male Fischer-344 rats received po doses of DNT daily for 5 days at 14, 35, and 70 mg/kg/day. Hepatic oxygen-insensitive cytosolic azoreductase activity was increased and microsomal nitroreductase was decreased by DNT treatments. A small but significant increase in liver/body weight ratio and in hepatic cytochromes P-450 and b₅ occurred in the absence of changes in microsomal biphenyl hydroxylase or aryl hydrocarbon hydroxylase activities. The patterns of in vitro microsomal metabolism of DNT were dependent on oxygen tension: under aerobic conditions, 2,4-dinitrobenzyl alcohol (DNBAlc) was the major metabolite whereas under anaerobic conditions no DNBAlc was detected; 2-amino-4-nitrotoluene (2A4NT) and 4-amino-2-nitrotoluene (4A2NT) were the major metabolites. Pretreatment of rats with phenobarbital or Aroclor 1254 increased the metabolism of 2,4-DNT to DNBAlc by six- to sevenfold. Metabolism to the alcohol was inhibited by SKF-525A. These data suggested that oxidative metabolism of 2,4-DNT to DNBAlc was mediated by cytochrome P-450-dependent mixed-function oxidases. Covalent binding studies showed that a maximum of only 7 pmol of 2,4-DNT-derived radioactivity was bound per milligram of microsomal protein per hour; this binding was increased to 1.0 nmol bound/mg protein/hr in microsomes from phenobarbital of Aroclor 1254-pretreated rats. It is concluded that 2,4-DNT treatment had little effect on the activity of some hepatic xenobiotic metabolizing enzymes and was readily metabolized by liver preparations in vitro. The pathways of in vitro metabolism were dependent on oxygen tension. This in vitro metabolism produced mostly polar metabolites which did not bind appreciably to microsomal macromolecules.