Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout: in vivo induction

1. Rainbow trout were dosed with prochloraz by i.p. injection of sprayed food pellets. Cytochrome P-450, two P-450-dependent activities, and two conjugase activities were measured in vitro in microsomal or cytosolic fractions. 2. Prochloraz increased cytochrome P-450 in liver, intestine, and pyloric caeca: maximum response occurred at 30-100 mg/kg i.p. In cold conditions, this increase persisted for more than 8 days after injection. 3. Hepatic 7-ethoxycoumarin-O-dealkylase (ECOD) and 7-ethoxyresorufin-O-dealkylase (EROD) were inhibited by prochloraz except in one assay in warm water where they increased. In intestine and pyloric caeca, ECOD and EROD were not detected, even when cytochrome P-450 was increased. 4. UDP-glucuronosyltransferase (1-naphthol as substrate) was unchanged or inhibited after prochloraz dosing. 5. Glutathione-S-transferase (o-dinitrobenzene as substrate), was unchanged or inhibited by prochloraz. 6. The measured level of enzymic activities was the result of induction and inhibition by prochloraz residues. Variations in basal activities and perhaps in prochloraz interactions were due to temperature acclimatization.     

Selective induction of renal microsomal cytochrome P-450-linked monooxygenases by 1,1-dichloroethylene in mice

Intraperitoneal administration of a single dose of 1,1-dichloroethylene (DCE) to C57 B1/6N mice (125 mg/kg) caused a selective 6- to 10-fold increase in renal microsomal 7-ethoxyresorufin O-deethylase ( EROD ) and 7-ethoxycoumarin O-deethylase ( ECOD ), without affecting benzo[a]pyrene hydroxylase activity (AHH) or total microsomal cytochrome P-450 content. The observed increases did not result from in vitro activation of the enzymes or from any analytical artifact. Moreover, studies with actinomycin D and cycloheximide demonstrated that the increases resulted from de novo enzyme synthesis. Maximal enzyme induction was observed after a DCE dose of approximately 125 mg/kg, and the induced enzyme decayed rapidly, returning to control levels in about 3 days. Compared to female mice, male mice had higher basal levels of renal EROD and ECOD and were more responsive to the inductive effects of DCE; this correlated with corresponding differences in microsomal cytochrome P-450 levels. Starvation of mice for 24 or 48 hr increased renal EROD and ECOD activities in both male and female mice, but not the extent observed after DCE. The present results support the view of multiple renal cytochrome P-450 isozymes.     

Interaction of monocrotophos and its novel thion analogues with microsomal cytochrome P-450: in vivo and in vitro studies in rat.

The binding of monocrotophos (MCP) and its two thion analogues (coded as RPR-II and RPR-V) to rat hepatic microsomal cytochrome P-450 (HMC) were investigated in vitro by difference spectroscopy. These three organophosphorus insecticides were found to bind stoichiometrically to HMC with very high affinity (Ks 34-50 microM). RPR-V showed the highest binding affinity followed by RPR-II and MCP. Association of these compounds with HMC occurred within 2 min of addition in the cuvette and therefore, appeared to be tight binding ligands of cytochrome P-450. In vivo studies at equitoxic doses of the three compounds 24 h after oral treatment in rats revealed that they all caused reduction in MC content in liver, lung, kidney and brain, as against induction in cardiac and splenic cytochrome P-450. These in vivo results suggest organ specificity in modulating the microsomal cytochrome P-450 (MC) content of hepatic and extra-hepatic tissues by the three compounds. Apparently, their binding affinity with HMC is strongly correlated with their LD50 value and has a substantial co-relationship with the cytochrome P-450 level in the liver.     

Effect of the suicide substrate 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine on the metabolism of xenobiotics and on cytochrome P-450 apoproteins

Treatment of rats with the cytochrome P-450 suicide substrate, 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), produced a 95% inhibition of the in vivo demethylation of either aminopyrine or morphine within 2 hr. One-carbon metabolism of formaldehyde or formate to carbon dioxide was not altered. DDEP also produced a time-dependent decrease in total hepatic microsomal cytochrome P-450 but had no effect on either NADPH-cytochrome c reductase or p-nitrophenol glucuronyl-transferase activities up to 24 hr after administration. A rapid decrease in rat liver microsomal aniline hydroxylation and ethoxyresorufin deethylation was observed in vitro following DDEP administration. Although in vitro testosterone metabolism to 16 alpha-, 16 beta-, and 2 alpha-hydroxy metabolites was depressed profoundly by DDEP in microsomes from untreated and 3-methylcholanthrene-treated animals, 7 alpha-hydroxylation of testosterone was much less affected. Immunochemical quantification of various microsomal cytochrome P-450 protein moieties showed that cytochromes P-450 beta NF-B, P-450UT-A, P-450PCN-E, and P-450PB-C were decreased in hepatic microsomes from DDEP-treated rats. However, the protein moiety of cytochrome P-450UT-H was not diminished and the immunoreactive protein for cytochromes P-450UT-F, P-450PB-B, and P-450ISF-G was only slightly decreased. These results show that DDEP treatment leads to marked decreases in holoprotein and apoproteins of many but not all hepatic microsomal cytochrome P-450 isozymes.     

Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout: inhibition in vitro and time course of induction in vivo

1. Interactions between the fungicide prochloraz and the hepatic cytochrome P-450 of rainbow trout were studied by determination of enzymic activities in vitro using the microsomal fraction, and by kinetic studies. 2. Prochloraz inhibited 7-ethoxyresorufin-O-deethylase (EROD) in vitro. This inhibition was partially non-competitive: the enzyme-substrate-inhibitor (ESI) complex was catalytically active (68% of the activity of the enzyme-substrate complex). 3. Prochloraz in vitro inhibited aldrin epoxidase (AE) by a linear mixed-type mechanism. This inhibition might be high because of high affinity for prochloraz and lack of catalytic activity of the ESI complex. 4. Effects of prochloraz in vivo were studied as a function of time after intraperitoneal injection. Total cytochrome P-450 increased for more than 21 days. 5. EROD increased slightly at day 4 and then returned to control level. Kinetics showed an increase in apparent Km and Vmax. AE was strongly inhibited at day 4 (large increase in apparent Km) and then returned to control level in 21 days. 6. Spectral interactions with aniline showed strong inhibition and recovery to an activated level at day 21.     

Effect of cytochrome P-450 and flavin-containing monooxygenase modifying factors on the in vitro metabolism of amiodarone by rat and rabbit

Experiments were conducted to affirm hepatic cytochrome P-450 involvement in the biotransformation of the class III antiarrhythmic agent, amiodarone (Am; Cordarone X) to its major metabolite, desethylamiodarone (DEA). Male Sprague-Dawley rats and male New Zealand white rabbits were treated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) (to induce cytochrome P-450 (PB-inducible cytochrome(s) P-450) or P-448 (MC-inducible cytochrome P-450). In vivo decreases in rat hepatic microsomal cytochrome P-450 were achieved either by a single ip dose of CCl4 or by a 2-day treatment with CoCl2. In vitro biotransformation of Am by hepatic microsomes from PB-induced and 3-MC-induced rats and PB-induced rabbits was significantly greater than that from noninduced animals. Conversely, in vitro DEA production was significantly decreased with hepatic microsomes from CCl4- and CoCl2-pretreated rats. The classic P-450 inhibitors, piperonyl butoxide, SKF 525A, n-octylamine, and CO provided a significant reduction in the in vitro formation of DEA by microsomes from induced animals. In vitro DEA formation by hepatic microsomes from PB- and 3-MC-induced rats was significantly decreased by 0.5 mM chloroquine (specific inhibitors of PB-inducible cytochrome(s) P-450) and 0.3 mM quinacrine (specific inhibitor of MC-inducible cytochrome(s) P-450), respectively. Further evidence for involvement of gut microsomal flavin-containing monooxygenase was provided by the inhibition of gut microsomal-mediated in vitro DEA formation in the presence of methimazole. Methimazole had no effect on hepatic microsomal DEA production in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effects of an imidazole antifungal, prochloraz, on spectral and catalytic properties of microsomal cytochromes P-450

1. The imidazole antifungal agent, prochloraz, elicited type II spectral interactions with microsomal cytochromes P-450 from rats pretreated with phenobarbital (PB), 3-methyl-cholanthrene (MC) and dexamethasone (DEX). 2. Prochloraz interacts strongly with type I binding sites of both PB- and DEX-induced cytochromes P-450 and to a lesser extent with cytochromes P-450 from MC-induced rats. 3. The antifungal derivative was a more potent inhibitor of the troleandomycin-nitrosoalkyl-cytochrome P-450 complex formation in DEX-induced microsomes than of the isosafrole-carbene-cytochrome P-450 complex formation in MC-pretreated rats. 4. Prochloraz is a strong inhibitor of the cytochrome P-450-dependent mixed-function oxidases in rat liver microsomal preparations.     

In vitro effects of an imidazole antifungal,prochloraz, on spectral and catalytic properties of microsomal cytochromes P-450

1. The imidazole antifungal agent, prochloraz, elicited type II spectral interactions with microsomal cytochromes P-450 from rats pretreated with phenobarbital (PB), 3-methyl-cholanthrene (MC) and dexamethasone (DEX).

2. Prochloraz interacts strongly with type I binding sites of both PB-and DEX-induced cytochromes P-450 and to a lesser extent with cytochromes P-450 from MC-induced rats.

3. The antifungal derivative was a more potent inhibitor of the troleandomycin-nitrosoalkyl-cytochrome P-450 complex formation in DEX-induced microsomes than of the isosafrole-carbene-cytochrome P-450 complex formation in MC-pretreated rats.

4. Prochloraz is a strong inhibitor of the cytochrome P-450-dependent mixed-function oxidases in rat liver microsomal preparations.     

Qualitative and quantitative changes in cytochrome P-450-dependent xenobiotic metabolism in pulmonary microsomes and isolated Clara cell populations derived from ozone-exposed rats

The effect of a prolonged ozone exposure (1.6 mg ozone/m3; 7 d; 24 h/d) on pulmonary cytochrome P-450-dependent xenobiotic metabolism was studied both in whole rat lung as well as in isolated bronchiolar Clara cell preparations. Ozone exposure was demonstrated to result in significant quantitative but also qualitative changes. All components of the pulmonary microsomal electron transport system appeared to be significantly increased in the lungs of exposed animals both per lung and per gram lung, although increases were no longer observed when expressed per milligram microsomal lung protein. Remarkably, it was demonstrated that the increases in the components of the pulmonary cytochrome P-450 system were not accompanied by a concomitant increase in all cytochrome P-450-dependent substrate conversions. In whole-lung microsomes ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities were unchanged or even significantly reduced when expressed per lung, per gram lung, per milligram microsomal protein, or per picomole cytochrome P-450. In contrast to these observations, pentoxyresorufin O-dealkylation appeared to be significantly increased upon ozone exposure when expressed per lung, per gram lung, and even per picomole cytochrome P-450. Clara cell populations isolated from ozone exposed rats showed a comparable qualitative shift in cytochrome P-450-dependent substrate conversion characteristics. On a cellular basis, ozone exposure resulted in a significant reduction of ethoxycoumarin and ethoxyresorufin O-deethylation and did not affect pentoxyresorufin O-dealkylase activity. Additional experiments, in which ozone-mediated inactivation of microsomal cytochrome P-450-dependent substrate metabolism was studied in vitro, demonstrated that the qualitative changes observed after in vivo exposure cannot be ascribed to a disproportional inactivation of different cytochrome P-450 isoenzymes. Based on these results and on lung morphometrics and cell isolation data presented, the observed effects should rather be ascribed to (1) the proliferation of cytochrome P-450 containing cell populations and (2) intrinsic cellular biochemical changes. The quantitative and qualitative ozone-induced changes in pulmonary cytochrome P-450-linked enzyme characteristics in whole lung and within specific lung cells, as demonstrated in the present study, may be expected to have important implications for the toxicity of xenobiotics whose (de)toxification depends on pulmonary cytochrome P-450-dependent metabolism.     

Bioavailability and cytochrome P-450 induction from 2,3,7,8-tetrachlorodibenzo-P-dioxin contaminated soils from Times Beach, Missouri, and Newark, New Jersey

Bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from contaminated soils from Times Beach, Missouri and Newark, New Jersey, was examined using liver concentrations and toxicity in guinea pigs observed up to 60 days following a single oral administration, and induction of cytochrome P-450 in rats sacrificed 24 hours after a single oral dose as endpoints. Both soils are contaminated with several chlorinated dioxins and numerous other compounds. Times Beach soil resulted in greater TCDD concentration in liver and TCDD was considerably more bioavailable from Times Beach soil than from Newark soil. However, both soils induced cytochrome P-450 activity to approximately the same extent. Moreover, similar banding patterns of microsomal proteins were seen on polyacrylamide electrophoretic gels. The many other compounds present in the soils, particularly in Newark, may account for the similar protein bands and levels of cytochrome P-450 observed.     

Studies on the cytochrome P-450 of avocado (Persea [corrected] americana) mesocarp microsomal fraction

1. Because of the low concentration of cytochrome P-450 in avocado fruit, microsomal fractions were prepared using polyethylene glycol aggregation and low-speed centrifugation, thus avoiding the need for high-speed centrifugation of large volumes of post-mitochondrial supernatant. Recoveries of cytochrome P-450 by this means (0.29 nmol/g tissue) were similar to those after the usual high-speed centrifugation preparation (0.26 nmol/g). The cytochrome P-450 content of tulip bulb (0.30 nmol/g) was similar to that of avocado, but both plant tissues had much lower P-450 contents than did rat liver (13.0 nmol/g). 2. Spectral studies indicate that cytochrome P-450 of avocado mesocarp microsomal fraction binds fewer substrates than does the rat liver enzyme system. Type I binding spectra are given by fatty acids (C7-C14), aryl hydrocarbons (C7-C12), p-chloro-N-methylaniline and N,N-dimethylaniline. Type II binding is seen with inhibitors of mammalian cytochrome P-450 such as metyrapone, and with the imidazole antifungal agents such as clotrimazole. 3. These binding spectra provide a rapid method for identifying possible substrates and inhibitors of avocado cytochrome P-450, and also provide information concerning the nature of the active site of avocado cytochrome P-450. 4. Avocado cytochrome P-450 catalysed the N-demethylation of N,N-dimethylaniline (17.1 nmol/min per nmol P-450) and p-chloro-N-methylaniline (13.1 nmol/min per nmol P-450), and the hydroxylation of lauric (dodecanoic) acid (1.1 nmol/min per nmol P-450).     

Cimetidine interaction with liver microsomes in vitro and in vivo. Involvement of an activated complex with cytochrome P-450

The O-deethylation of 7-ethoxycoumarin was inhibited in a mixed type manner by cimetidine in vitro and in microsomes isolated from rats treated with cimetidine in vivo. It was found that the inhibition was even greater if cimetidine was preincubated with the microsomal suspension in the presence of an NADPH-generating system prior to the addition of substrate. In vitro the decrease in activity was accompanied by a decrease in cytochrome P-450 content. This decrease was unaffected by the addition of EDTA to the microsomal suspensions, eliminating the possibility that free radical production was responsible for the decrease in cytochrome P-450. The decrease in activity and cytochrome P-450 content following preincubation of microsomal suspensions with cimetidine could be attenuated if potassium ferricyanide was added to the suspensions. The deethylation activity and cytochrome P-450 content of liver microsomes prepared from cimetidine-treated rats was decreased compared to control animals. The activity and cytochrome P-450 content of microsomes from cimetidine-treated rats could also be restored if microsomes were washed with potassium ferricyanide prior to incubation with substrate. It is proposed that an intermediate complex of cimetidine and cytochrome P-450 could be involved in the inhibition of microsomal metabolism by cimetidine.     

1-(8-Methoxy-4,8-dimethylnonyl)-4-(1-methyl-ethyl)benzene (MV-678): a reversible inducer of rat hepatic microsomal drug metabolism

MV-678 [1-(8-methoxy-4,8-dimethynonyl)-4-(1-methylethyl)benzene], a recently developed insect growth regulator, increased the hepatic cytochrome P-450-dependent monooxygenase enzymes that metabolize endogenous and exogenous chemicals. In an initial set of experiments, male and female rats received 0, 50, or 800 mg/kg X d of MV-678 by gavage for 3 d, and in a second set of experiments, male rats received 0, 50, or 800 mg/kg X d of MV-678 by gavage for 30 d. A significant increase in both absolute and relative liver weight, microsomal protein content, cytochrome P-450 content, NADPH-cytochrome P-450 reductase activity, and ethylmorphine N-demethylase activity was observed in male and female rats at the high dose level at 3 d. Similar increases were observed in the 800-mg/kg X d males at 30 d. Hepatocellular hypertrophy and proliferation of endoplasmic reticulum observed at both 3 and 30 d correspond to and was consistent with microsomal enzyme induction. Reversibility of both induction and changes in morphology was determined by measuring the same parameters in animals treated for 30 d after a 15- or 30-d recovery period. At 15 d recovery, all biochemical parameters at the high dose level, except relative liver weight and microsomal ethylmorphine N-demethylase activity, had returned to control levels. No significant differences between the control and high dose group animals were noted at 30 d recovery. The hepatocellular changes observed in the high-dose group at 30 d were less apparent at 15 d recovery, and absent at 30 d recovery.     

Induction of P-450 cytochromes 2E2, 1A1, and 1A2 by imidazole in neonatal rabbits.

Cytochrome P-450 2E1 is induced in adult rabbits by treatment with alcohol, imidazole, and a variety of other agents, as shown earlier in this laboratory, but it is not known whether the highly homologous P-450 2E2 is similarly induced. In this study, the effects of imidazole on 2E2 expression were examined in neonatal rabbits, in which 2E1 is not detectable. Treatment of the animals with imidazole on days 8 through 11 after birth caused a 3-fold increase in the content of total P-450 in liver microsomes. In contrast, the microsomal content of cytochrome b5 and NADPH-P450 reductase was not changed. Immunoblot analysis revealed a significant increase in the level of P-450 2E2 (3-fold) as well as 1A1 (> 10-fold) and 1A2 (> 2-fold) in hepatic microsomes from imidazole-treated neonatal rabbits. The rates of microsomal N-demethylation of N-nitrosodimethylamine and O-deethylation of 7-ethoxyresorufin were similarly increased from 1.3 and 0.03 nmol/min/mg protein, respectively, to 5.6 and 0.24 nmol/min/mg protein, respectively, by imidazole treatment. Blot analysis indicated that the levels of 2E2, 1A1, and 1A2 mRNAs are not increased by imidazole treatment and that 2E1 mRNA is not detectable in either untreated or imidazole-treated neonates. The induction of P-450 2E2 was confirmed by NH2-terminal amino acid sequence analysis of immunopurified 2E protein from hepatic microsomes of imidazole-treated neonatal rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical toxicology of argemone oil. I. Effect on hepatic cytochrome P-450 and xenobiotic metabolizing enzymes

The in vivo effect of argemone oil on hepatic xenobiotic metabolizing enzymes was investigated in albino rats following either a single (10 ml kg-1 body wt.) or multiple intraparenteral doses (5 ml kg-1 body wt.) for three days. Animals sacrificed 72 h after a single intraparenteral dose of argemone oil exhibited a significant loss of hepatic cytochrome P-450 (35%) and cytochrome b5 (34%) contents and inhibition of aminopyrine-N-demethylase (APD), aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin-O-deethylase (ECD) activities (21-39%). Three successive 24-hourly intraparenteral injections of argemone oil followed by sacrificing the animals after 24 h of the last injection, showed a greater degree of inhibition of the content of cytochrome P-450 (58%) and its dependent mixed-function oxidases (35-63%). Also, multiple treatment of argemone oil caused a depletion of endogenous hepatic glutathione (GSH) content (72%) with a concomitant increase in lipid peroxidation (177%) and decrease in glutathione-S-transferase (GST) activity (30%). A significant decrease in relative liver weight (39%) was observed in animals treated with multiple treatment of argemone oil. These results suggest that argemone oil can alter both membrane and cytosolic defences and destabilizes the hepatic cytochrome P-450 dependent mixed-function oxidase system, so that it tips in the direction of autooxidative peroxidation of lipids.     

Induction and inhibition of rat hepatic drug metabolism by N-substituted imidazole drugs

Three daily administrations of N-substituted imidazole antimycotics, clotrimazole (CloTZ, 75 mg/kg/day), miconazole (MCZ, 150 mg/kg/day), or tioconazole (TCZ, 150 mg/kg/day), but not the 4,5-disubstituted imidazole cimetidine (350 mg/kg/day) or imidazole (200 mg/kg/day for 4 days), induced rat hepatic cytochrome P-450 and other drug-metabolizing enzymes. These findings paralleled in vitro observations where CloTZ, MCZ, and TCZ were several orders of magnitude more potent as inhibitors of p-nitroanisole O-demethylase activity in control male rat liver microsomes than cimetidine or imidazole. Although no marked difference in inhibitory potency was evident among the N-substituted imidazoles, there were qualitative and quantitative differences in the profiles and extents of induction of various cytochrome P-450-dependent monooxygenases and Phase II conjugation enzymes. Cytochrome P-450 was elevated dramatically by CloTZ (3-4 times the control) and to a lesser extent by MCZ and TCZ (congruent to 1.5 times the control). For all agents, there was an increase in metyrapone binding approximately equivalent to the additional (i.e. above control) cytochrome P-450. Despite the large difference in cytochrome P-450 induction by CloTZ, MCZ, and TCZ, these agents elevated p-nitroanisole demethylase and aniline hydroxylase to similar extents (3-5 X and 1-2 X control, respectively). All agents induced erythromycin and ethylmorphine demethylation in proportion to cytochrome P-450. Ethoxyresorufin O-de-ethylation was not substantially affected by any agent. Large differences in the extent and specificity of induction of microsomal glucuronide conjugations were also evident.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of a cytochrome P-450-containing mixed-function oxidase system in the pond snail, Lymnaea stagnalis

1. The occurrence of an as yet unidentified cytochrome P-450 in the microsomal fraction of the digestive gland of the snail, Lymnaea stagnalis was studied. 2. Studies in vivo and in vitro (digestive gland homogenates or the 170,000g fraction) of the cytochrome P-450-mediated metabolism of substrates such as biphenyl, pentoxy- and ethoxy-resorufin and aminopyrine have been made. 3. Cytochrome P-450 concentration in the digestive gland calculated from CO-difference spectra was 0.30 +/- 0.05 nmol/g tissue. This amount was not increased either by phenobarbital or by 3-methylcholanthrene. 4. Aniline binding spectra resembled normal type II spectra, while type I model substrates such as hexobarbital and 2,2'-dichlorobiphenyl showed type II- or reversed type I-like spectra. 5. O-Deethylation of 7-ethoxyresorufin did not occur, but 7-pentoxyresorufin O-depentylation activity (80.4 +/- 28.6 pmol resorufin/g per hour) and aminopyrine N-demethylation activity (375 +/- 96 pmol formaldehyde/g per minute) were demonstrated. 6. 4-Hydroxybiphenyl was the major metabolite of biphenyl, while minor amounts of 2-hydroxybiphenyl were formed (in vivo: 63.7 nmol 4-hydroxybiphenyl and 3.33 nmol 2-hydroxybiphenyl per snail per 24 h, after an oral dose of 778.2 nmol biphenyl; in vitro 118 +/- 21 pmol and 21 +/- 9 nmol, respectively (digestive gland homogenate/mg protein, per hour). 7. The results indicate that the isoenzymes involved in the observed MFO activities resemble isoenzymes P-450b/e.     

Effect of 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl on the induction of hepatic lipid peroxidation and cytochrome P-450 associated enzyme activities in rats

Polychlorinated biphenyls (PCBs) are environmental contaminants that have been widely used for various industrial purposes. In spite of numerous studies on PCBs, however, their mechanism of toxicity remains unknown. The role of cytochrome P-450 in PCBs induced hepatic lipid peroxidation is controversial. Therefore, the present study was undertaken to study the mechanism of action of two PCBs and their role in cytochrome P-450 induction and lipid peroxidation, determined in vivo and during the incubation of subcellular fractions. We also examined whether agonist/antagonist activities between the two PCBs were occurring. Two PCBs were studied: 3,3',4,4'-tetrachlorobiphenyl (PCB-77), a non-ortho-substituted, coplanar PCB; and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153), a di-ortho-substituted, non-planar PCB. Groups of male Sprague-Dawley rats were given a single i.p. injection of one of the two PCBs (at doses of 30, 150, or 300 micromol/kg), both PCBs (at doses of 30 or 150 micromol/kg), or vehicle alone. Rats were sacrificed after 2, 6, or 24 h; or 2, 6, or 10 days. Cytochrome P-450 induction occurred as early as 2 h with PCB-77 and 24 h with PCB-153. Significant increases in thiobarbituric acid reactive substances (TBARS) content in liver tissue occurred 2, 6 and 10 days after treatment with PCB-77 and PCB-153; it was unclear whether these PCBs were synergistic in their induction of TBARS formation. Liver microsomal fractions incubated with NADPH only showed increased TBARS formation at the highest doses of PCB-77 and PCB-153 after 6 days. The results indicate that both PCBs induced cytochrome P-450 enzymes and enhanced lipid peroxidation in liver and subcellular fractions but with different potencies and onsets of action. The results also indicate a larger time difference between cytochrome P-450 induction and lipid peroxidation for PCB-77. Thus, both PCB-77 and PCB-153 are toxic to cells, but may act via different mechanisms to induce their effects.     

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.