Hepatic P450 Activity Research Articles

Proteins combination on PHBV microsphere scaffold to regulate Hep3B cells activity and functionality: A model of liver tissue engineering system

The synergistic effects of extracellular matrix (ECM) protein combinations on Hep3B cell proliferation and functions are studied herein. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres were covalently conjugated with three types of proteins, collagen (type I), laminin, and fibronectin, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide cross linkers. Successful conjugations of protein molecules were verified by the presence of nitrogen peaks in X-ray photoelectron spectroscopy. The densities of grafted proteins were quantified using Micro-BCA kit. A human hepatoma cell line, Hep3B, was then cultured in vitro on the ECM proteins-modified microspheres for 2 weeks. Cell proliferation was estimated using MTT method, and two hepatic functions, albumin secretion and P-450 activity, were evaluated using ELISA and EROD assays, respectively. The results indicated that combination of the three ECM proteins on microsphere surfaces has a significant effect on the proliferation of Hep3B cells, thus better mimicking the in vivo environment for liver tissue engineering.

Ginsenoside Metabolites, Rather Than Naturally Occurring Ginsenosides, Lead to Inhibition of Human Cytochrome P450 Enzymes

There is still an argument about ginseng-prescription drug interactions. To evaluate the influence on cytochrome P450 (P450) activities of ginseng in the present study, the influence on P450 activities of naturally occurring ginsenosides and their degradation products in human gut lumen was assayed by using human liver microsomes and cDNA-expressed CYP3A4. The results showed that the naturally occurring ginsenosides exhibited no inhibition or weak inhibition against human CYP3A4, CYP2D6, CYP2C9, CYP2A6, or CYP1A2 activities; however, their main intestinal metabolites demonstrated a wide range of inhibition of the P450-mediated metabolism. There was no mechanism-based inhibition found on these P450 isoforms. It is noteworthy that Compound K, protopanaxadiol (Ppd), and protopanaxatriol (Ppt) all exhibited moderate inhibition against CYP2C9 activity, and Ppd and Ppt also exhibited potent competitive inhibition against CYP3A4 activity. We suggest that after oral administration, naturally occurring ginsenosides might influence hepatic P450 activity in vivo via their intestinal metabolites.

Impact of Andrographis paniculata crude extract on mouse hepatic cytochrome P450 enzymes

Modulatory influence of Andrographis paniculata crude extract on cytochrome P450 (CYP) enzymes was performed by administration of the crude extract of Andrographis paniculata to ICR male mice. Total hepatic P450 content was not significantly modified by either the aqueous or the alcoholic extracts of Andrographis paniculata. Assessment of hepatic microsomal P450 activities by alkoxyresorufin O-dealkylations noted that both the aqueous and alcoholic extracts of Andrographis paniculata significantly increased ethoxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase activities, while those of methoxyresorufin O-dealkylase activities were not elevated. These results suggested that Andrographis paniculata might effectuate hepatic cytochrome P450 enzymes of which CYP1A1 and CYP2B are the responsive P450 isoforms.

PHARMACOKINETICS AND DISPOSITION OF THE KAVALACTONE KAWAIN: INTERACTION WITH KAVA EXTRACT AND KAVALACTONES IN VIVO AND IN VITRO

Reported adverse drug interactions with the popular herb kava have spurred investigation of the mechanisms by which kava could mediate these effects. In vivo and in vitro experiments were conducted to examine the effects of kava extract and individual kavalactones on cytochrome P450 (P450) and P-glycoprotein activity. The oral pharmacokinetics of the kavalactone, kawain (100 mg/kg), were determined in rats with and without coadministration of kava extract (256 mg/kg) to study the effect of the extract on drug disposition. Kawain was well absorbed, with >90% of the dose eliminated within 72 h, chiefly in urine. Compared with kawain alone, coadministration with kava extract caused a tripling of kawain AUC(0-8 h) and a doubling of C(max). However, a 7-day pretreatment with kava extract (256 mg /kg/day) had no effect on the pharmacokinetics of kawain administered on day 8. The 7-day pretreatment with kava extract only modestly induced hepatic P450 activities. The human hepatic microsomal P450s most strongly inhibited by kava extract (CYP2C9, CYP2C19, CYP2D6, CYP3A4) were inhibited to the same degree by a "composite" kava formulation composed of the six major kavalactones contained in the extract. K(i) values for the inhibition of CYP2C9 and CYP2C19 activities by methysticin, dihydromethysticin, and desmethoxyyangonin ranged from 5 to 10 microM. Kava extract and kavalactones (< or =9 microM) modestly stimulated P-glycoprotein ATPase activities. Taken together, the data indicate that kava can cause adverse drug reactions via inhibition of drug metabolism.

Effects of serum from patients with chronic renal failure on rat hepatic cytochrome P450

1. In humans, chronic renal failure (CRF) is associated with decreased hepatic drug metabolism, particularly that mediated by the cytochrome P450 (P450). The mechanisms remain poorly understood. The present study aimed to investigate the effects of the serum of patients with CRF on liver P450, and to evaluate whether renal replacement therapies (dialysis or transplantation) impede the inhibition of CRF serum on P450. 2. Rat hepatocytes were incubated for 24 h with serum from patients with severe CRF and from controls to measure (1) P450 level, (2) protein expression and mRNA levels of P450 isoforms and (3) metabolic activities of CYP3A and CYP1A. Similar experiments were performed with serum of patients once on chronic hemodialysis and after kidney transplantation. 3. In rat hepatocytes incubated for 24 h with serum from patients with CRF, P450 level and protein expression, as well as mRNA levels of P450 isoforms (CYP1A2, 2C6, 2C11, 2D1/2D2, 3A2 and 4A1/4A3), were decreased by more than 45% (P<0.001) compared to control serum, while the levels of CYP2E1 were not modified. CYP3A and CYP1A activities were decreased by 51 and 59% (P<0.001), respectively. The inhibitory effect of serum obtained from patients before first dialysis was similar after 1 or 6 months on chronic hemodialysis but was lost after successful kidney transplantation. In CRF serum, the fraction containing proteins between 10 and 15 kDa decreases P450. 4. Human uremic serum contains mediator(s) that decreases rat hepatic P450 activity and expression secondary to reduced gene expression. The inhibitory effect of serum persists even after initiation of dialysis, but disappears after normalization of renal function following kidney transplantation.

CYP3A4 and P-Glycoprotein Activity in Healthy Controls and Transplant Patients on Cyclosporin vs. Tacrolimus vs. Sirolimus

This study aimed to determine the impact of maintenance immunosuppressive therapy with cyclosporin A (CsA), tacrolimus (FK506) and sirolimus (Rapa) on the in vivo activity of both intestinal and hepatic cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (PGP) in renal transplant patients. The activity of these four elimination pathways was measured by the recently validated intravenous (iv) and per oral (po)14C erythromycin breath and urine test. In addition, overall hepatic P450 activity was measured by the 13C aminopyrin breath test. Three groups of stable renal transplant patients on maintenance therapy with corticosteroids (CS) and mycophenolate mofetil (MMF) plus either CsA or FK506 or Rapa were examined. A significant increase in intestinal CYP3A4 activity and a significant decrease in hepatic and intestinal PGP activity was seen in patients on CsA in comparison with those on FK506 or Rapa (p < 0.01). A similar analysis in six healthy volunteers at baseline and after intake of CsA, FK506 and Rapa confirmed the results seen in the patients. There was no difference in CYP3A4 and PGP activity in the patients taking either FK506 or Rapa and healthy controls. These data suggest that a different pattern of drug interactions might be expected in patients treated with CsA vs. FK506/Rapa.

The effects of albendazole and its metabolites on hepatic cytochromes P450 activities in mouflon and rat

Albendazole (ABZ) is a benzimidazole anthelmintic widely used in veterinary medicine. The effects of ABZ on cytochromes P450 were investigated in primary cultures of mouflon ( Ovis musimon) and rat ( Rattus norvegicus) hepatocytes. Besides ABZ, its two main metabolites (albendazole-sulphoxide, ABZSO and albendazole-sulphone, ABZSOO) were tested to clarify which compound is responsible for the induction potency of this benzimidazole drug. After 48 h incubation of hepatocytes with benzimidazoles (0.2–25 μM), ethoxyresorufin O-deethylation (EROD) and benzoxyresorufin O-dearylation (BROD) were measured and the P4501A and 3A protein levels were determined by Western blotting. All benzimidazoles provoked a significant increase of EROD and BROD activities in rat hepatocytes. ABZSO and ABZSOO seemed to be responsible for the induction effect of ABZ on P450s in rat. In mouflon, no pharmacologically significant induction of EROD and BROD activities by benzimidazoles tested was observed. From this point of view, anthelmintic therapy of mouflons with ABZ seems to be safe.

Inactivation of the Hepatic Cytochrome P450 System by Conditional Deletion of Hepatic Cytochrome P450 Reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.

Suppression of hepatic CYP3A1/2 and CYP2C11 by cyclosporine is not mediated by altering growth hormone levels.

Cyclosporine (CsA) suppresses drug metabolism by decreasing cytochrome P450 (P450) enzyme levels in rat liver. Growth hormone (GH) is known to pretranslationally regulate P450 expression. Thus, the suppression of P450 by CsA may involve GH as an intermediate. To address this question, we examined the effects of administering exogenous GH via twice daily subcutaneous injections and in conjunction with chronic subcutaneous CsA administration for 14 days on hepatic P450 expression. CsA alone decreased CYP3A1/2 and CYP2C11 significantly, in a manner similar to that previously found. When administered in the absence of CsA, GH also suppressed CYP3A1/2 and CYP2C11 protein levels as compared with GH vehicle. In the presence of CsA, GH did not cause further suppression of either CYP3A1/2 or CYP2C11 expression when compared with CsA treatment with GH vehicle. Testosterone in vitro catalytic assays confirmed that CsA and GH separately cause significant decreases in activity levels. Also, the concomitant administration of GH and CsA caused lowered production of 16alpha-, 2alpha-, 6beta-, and 2beta-hydroxytestosterone as compared with the administration of GH with CsA vehicle and as compared with the administration of GH vehicle with CsA. This study shows that GH is a dominating factor over CsA in determining hepatic P450 expression and activity. In addition, CsA does not seem to alter GH levels as a mediating event in suppressing P450 expression and activity. Since CsA given in combination with GH further suppressed P450 activity as compared with CsA given in combination with vehicle, this suggests that changes in hormonal status are likely to be one of the many factors that is responsible for the lack of a clear association between cyclosporine dosing and markers of toxicity.

The Influence of l-Glutamine on the Depression of Hepatic Cytochrome P450 Activity in Male Rats Caused by Total Parenteral Nutrition

Total parenteral nutrition (TPN) bypasses the gut leading to intestinal and hepatic dysfunction, including decreased hepatic cytochrome P450 (P450) activity. Glutamine prevents the TPN-associated changes in gut function and morphology. This study examined the effect of glutamine supplementation on hepatic P450 activities in male Sprague-Dawley rats receiving continuous TPN. Animals received continuous lipid-free TPN for 7 days with 0, 0.1, or 4.5% glutamine. Surgical controls were allowed free access to rat chow. The V(max)/K(m) ratios (intrinsic clearance) for the formation of 4-hydroxymidazolam (CYP3A) were 12.8, 14.6, and 27.7 microl/min/mg for TPN treatment with 0, 0.1%, or 4.5% glutamine, respectively, compared with a chow-fed control (37.1 microl/min/mg). The corresponding values for 1'-hydroxymidazolam formation (CYP3A) were 3.7, 6.1, 11.7, and 15.2 microl/min/mg, respectively. The addition of glutamine to TPN similarly affected the formation rates for 2beta- and 6beta-hydroxytestosterone (CYP3A), and these metabolite formation rates were highly correlated (r = 0.865; p < 0.001). The formation rates for 2alpha- and 16alpha-hydroxytestosterone (CYP2C) were also highly correlated (r = 0.892; p < 0.001). Parenteral glutamine modified the TPN-associated suppression of CYP3A and CYP2C activities in adult male rats receiving TPN.

Sex Difference in the Daily Rhythm of Hepatic P450 Monooxygenase Activities in Rats Is Regulated by Growth Hormone Release

Daily fluctuations have been observed in the hepatic cytochrome P450 monooxygenase activities of rodents. It was confirmed in our previous study that the P450 activities assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) showed obvious daily fluctuations in male rats with high values during the dark period. However, there is still very little information about the daily fluctuation of P450 activities in female rats. In the first experiment of the present study, the daily fluctuation of ACD activities in female rats was examined. The results indicated the absence of any apparent daily fluctuation in the hepatic ACD activities of the females, so the study confirmed a sex difference in the daily rhythm of the ACD activities. In the second experiment using hypophysectomized (Hpx) rats, it was investigated whether this sex difference in daily rhythm was attributable to growth hormone (GH) release, an activity which is known to affect the sex difference in the expression of P450 isoforms. It was found that the Hpx rats, given subcutaneous injections of GH twice during the light period to mimic a male pattern of GH secretion, showed obvious light–dark fluctuation in ACD activities with high values in the dark period, and similar results were obtained in sham-operated males. Conversely, the Hpx rats given continuous infusion of GH to mimic a female pattern of GH secretion did not show the light–dark fluctuation in ACD activities, and similar results were obtained in sham-operated females. In conclusion, there is a sex difference in the daily rhythm of the hepatic P450 activities in rats and this difference is apparently influenced by the difference in the patterns of GH secretions.

P450 Gene Induction by Structurally Diverse Xenochemicals: Central Role of Nuclear Receptors CAR, PXR, and PPAR

The biochemistry of foreign compound metabolism and the roles played by individual cytochrome P450 (CYP) enzymes in drug metabolism and in the toxification and detoxification of xenochemicals prevalent in the environment are important areas of molecular pharmacology and toxicology that have been widely studied over the past decade. Important advances in our understanding of the mechanisms through which foreign chemicals impact on these P450-dependent metabolic processes have been made during the past 2 years with several key discoveries relating to the mechanisms through which xenochemicals induce the expression of hepatic P450 enzymes. Roles for three “orphan” nuclear receptor superfamily members, designated CAR, PXR, and PPAR, in respectively mediating the induction of hepatic P450s belonging to families CYP2, CYP3, and CYP4 in response to the prototypical inducers phenobarbital (CAR), pregnenolone 16α-carbonitrile and rifampicin (PXR), and clofibric acid (PPAR) have now been established. Two other nuclear receptors, designated LXR and FXR, which are respectively activated by oxysterols and bile acids, also play a role in liver P450 expression, in this case regulation of P450 cholesterol 7α-hydroxylase, a key enzyme of bile acid biosynthesis. All five P450-regulatory nuclear receptors belong to the same nuclear receptor gene family (family NR1), share a common heterodimerization partner, retinoid X-receptor (RXR), and are subject to cross-talk interactions with other nuclear receptors and with a broad range of other intracellular signaling pathways, including those activated by certain cytokines and growth factors. Endogenous ligands of each of those nuclear receptors have been identified and physiological receptor functions are emerging, leading to the proposal that these receptors may primarily serve to modulate hepatic P450 activity in response to endogenous dietary or hormonal stimuli. Accordingly, P450 induction by xenobiotics may in some cases lead to a perturbation of endogenous regulatory circuits with associated pathophysiological consequences.

Note on assay of hepatic P-450 activity for evaluation of the function of bioartificial liver support system

Activity of cytochrome P-450 (cyt. P450) is often measured to evaluate the function of liver cells in bioartificial liver. Lidocaine is often employed as a substrate, and the amount of lidocaine remaining in the medium is analyzed by either gas chromatography (GC) or highperformance liquid chromatography (HPLC) after extraction. By GC analysis, the recovery of lidocaine was about 85% after extraction. This result suggests that the activity of cyt. P450 is estimated to be higher than the actual level if such a loss in recovery is not considered. It was also observed that albumin in the medium seriously affected the cyt. P450 activity assay, when lidocaine was at a high concentration in the medium. These points should be noted in the study of bioartificial liver. It was also shown that the extraction problem mentioned above could be overcome by directly injecting medium onto HPLC.

In vitro inhibition of hepatic cytochrome P450 and enzyme activity by butyltin compounds in marine mammals

The present study attempted to examine the in-vitro inhibition of hepatic microsomal P450 content and activity by butyltins in marine mammals and discussed on their possible effects in animals in the wild. Decreases in P450 content and the activities of ethoxyresorufin O-deethylase (EROD, catalyzed by CYPIA subfamily) and penthoxyresorufin O-depentylase (PROD, catalyzed by CYP2B subfamily) by tributyltin (TBT) were observed in in-vitro experiments using hepatic microsomes of a pinniped and a cetacean. Among P450 family, EROD activity is more sensitive to TBT than P450 content and PROD activity, indicating a specific mode of action of TBT on different P450 forms. On the other hand, dibutyltin and monobutyltin have no inhibitory effect on EROD activity at concentrations less than 0.5 m m, indicating that the inhibition of enzyme activity in hepatic microsome of marine mammal is mainly by TBT. TBT concentrations that affect P450 contents and activities are above 10 times higher than the values found in the liver of various marine mammals.

Do Endogenous Volatile Organic Chemicals Measured in Breath Reflect and Maintain CYP2E1 Levelsin Vivo?

The effect oftrans-1,2-dichloroethylene (DCE), an inhibitor of cytochrome P450 (P450) 2E1 (CYP2E1), on the composition and quantity of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with hepatic P450 activity and content. Hepatic microsomes were prepared from groups of rats prior to dosing and at 2, 5, 12, and 24 hr postdosing with DCE (100 mg/kg ip), and total P450 content and the activity of CYP2E1 was determined. Breath was collected from parallel groups of rats predose and at several intervals that encompassed the time points for rats euthanized for microsome preparation. Over 100 breath components were identified by GC/MS and quantitated by GC/FID. The overall change in the profile of breath VOCs resulting from administration of DCE was striking. An increase of approximately 1000% was measured in the mass of non-DCE-derived VOCs exhaled 4–6 hr after dosing, but there was no increase in hepatic lipid peroxidation. In addition to hexane, short-chain methyl ketones were particularly affected, and percentage increases in response to inhibition were inversely related to chain length, with acetone and 2-butanone > 2-pentanone ⪢ 2-hexanone > 2-heptanone. There were no statistically significant decreases in total content of P450, but the activity of CYP2E1 was diminished about 65% at 2 and 5 hr after DCE treatment. However, 24 hr after inhibitor administration the total mass of VOCs expired was only marginally elevated above baseline and CYP2E1 activity was not significantly different from that of untreated rats. The compounds most markedly increased upon inhibition of CYP2E1 are also excellent inducers of that isozyme, and this finding is consistent with the hypothesis that these chemicals are important to the normal homeostasis of CYP2E1. The increase in breath components observed following inhibition of CYP2E1 suggests that VOCs in breath can reflect the activity of that isozymein vivo.

Gadolinium chloride reduces cytochrome P450: Relevance to chemical-induced hepatotoxicity

The Kupffer cell inhibitor, gadolinium chloride (GdCl3), protects the liver from a number of toxicants that require biotransformation to elicit toxicity (i.e. 1,2-dichlorobenzene and CCl4), as well as compounds that do not (i.e. cadmium chloride and beryllium sulfate). The mechanism of this protection is thought to result from reduced secretion of inflammatory and cytotoxic products from Kupffer cells (KC). However, since other lanthanides have been shown to decrease cytochrome P450 (P450) activity, the following studies were designed to determine if GdCl3 pretreatment alters hepatic P450 levels or activity. The toxicological relevance of GdCl3-mediated alterations in P450 activity was also estimated by determining the effect of GdCl3 pretreatment on the susceptibility of primary cultured hepatocytes to CCl4 and cadmium chloride (CdCl2). Male and female Sprague-Dawley rats were given GdCl3 (i.v., 10 mg/kg). Twenty-four hours later, livers were either processed for preparation of microsomes or for primary cultures of hepatocytes. Gadolinium chloride treatment reduced total hepatic microsomal P450 as well as aniline hydroxylase activity by approximately 30% in males and 20% in females. In hepatocytes isolated from rats pretreated with GdCl3, the toxicity caused by CCl4, but not CdCl2 was reduced. Interestingly, when GdCl3 was administered in vitro to microsomes, there was no effect on either the microsomal P450 difference spectra or p-hydroxylation of aniline. However, when GdCl3 was incubated with isolated hepatocytes, the cytotoxicity of CCl4 (but not CdCl2) was partially attenuated. These results suggest that, in addition to its inhibitory effects on KC, GdCl3 produces other effects which may alter the susceptibility of hepatocytes to toxicity caused by certain chemicals.

Regulation of Cytochromes P450 During Inflammation and Infection

Hepatic P450 activities are profoundly affected by various infectious and inflammatory stimuli, and this has clinical and toxicological consequences. Whereas the expression of most P450s in the liver is suppressed, some are induced. Many of the effects observed in vivo can be mimicked by pro-inflammatory cytokines and IFNs, and P450s are differentially regulated by these agents. Therefore, different cytokine profiles and concentrations in the vicinity of the hepatocyte in different models of inflammation may result in qualitatively and quantitatively different effects on populations of P450s. In addition to cytokines, glucocorticoids may have an important role in P450 regulation in stress conditions, including that caused by inflammatory stimuli. Although in many cases the decreases in activity are due primarily to a down-regulation of P450 gene transcription, it is likely that modulation of RNA and protein turnover, as well as enzyme inhibition, contributes to some of the observed effects. The mechanisms whereby these effects are produced may also vary with both the P450 under study and the time course of the effect. The complexity of the P450 response to inflammation and infection means that all of the above factors must be considered when trying to predict the effect of a given infectious or inflammatory condition on the clinical or toxic response of humans or animals to an administered drug or toxin. The question of whether the down-regulation of the hepatic P450 system to inflammation or infection is a homeostatic or pathological response cannot be answered at present. It is difficult to discern the physiological benefit of reducing hepatic P450 activities, unless it is to prevent the generation of reactive oxygen species generated by uncoupled catalytic turnover of the enzymes. On the other hand, as we proposed some years ago [64], the suppression of P450 may be due to the liver's need to utilize its transcriptional machinery and energy for the synthesis of APPs involved in the inflammatory response. In that case, one could ask why the organism has gone to the trouble of employing differential mechanisms for suppression of P450. One answer could be that the response evolved after the divergence of many of the P450 genes, necessitating the evolution of multiple redundant mechanisms for P450 suppression. In contrast to the down-regulation of P450s in the liver, the induction of several forms in this and other tissues suggests a more specific homeostatic role of these effects, e.g., in generation or catabolism of bioactive metabolites.

Congener specific accumulation and toxic assessment of polychlorinated biphenyls in common cormorants, Phalacrocorax carbo, from Lake Biwa, Japan

Isomer-specific accumulation of polychlorinated biphenyls (PCBs) including di-, mono- and non- ortho congeners and hepatic P-450 activities were determined in adult common cormorants from Lake Biwa, Japan. The mean total PCB levels in male and female birds were 7.2 ± 6.1 and 2.1 ± 0.74 μg g −1 wet wt, respectively, in the liver. The highly biomagnified congeners were IUPAC 126, 153, 169, 180 and 194, whereas a higher degree of biotransformation could be observed in both meta-para chlorine unsubstituted congeners in the cormorant liver. The estimated metabolic index also showed that common cormorants had higher PB-type enzyme activities than some avian and marine mammals but poor MC-type enzyme activities. The concentrations of non- ortho coplanar congeners were in the order of IUPAC 126 > IUPAC 169 > IUPAC 77 with mean values 6.1 ± 5.9, 1.3 ± 1.4 and 0.43 ± 0.26 ng g −1 wet wt, respectively. The calculated mean 2,3,7,8-TCDD toxic equivalent (TEQ) concentration in cormorants was 1.8 ± 1.7 ng g −1 wet wt and was dominated by IUPAC 118, followed by IUPAC 126. A significant increase of ethoxyresorufin- O-deethylase (EROD) and pentoxyresorufin- O-deethylase (PROD) activities was observed with estimated TEQ of PCBs in the cormorants, suggesting that the current contamination level is sufficient for altering their biochemical responses.

Comparative Short-Term Effects of Methyl Tertiary Butyl Ether and Unleaded Gasoline Vapor in Female B6C3F1 Mice

PS-6 unleaded gasoline (UG) and methyltert-butyl ether (MTBE), an UG additive, with long-term exposure at high concentrations increased liver tumors selectively in female mice. PS-6 UG is a liver tumor promoter inN-nitrosodiethylamine-initiated female mice and produces short-term effects potentially relevant to its tumor promoting ability. The new formulation of UG (91-01) and MTBE were evaluated for similar short-term effects in mouse liver. Mice were exposed to 7814 ppm MTBE, 2014 ppm 91-01 UG, or 2028 ppm PS-6 UG vapor for 3 or 21 days, 6 hr/day, 5 days/week. Relative liver weights increased and uterine weights decreased in MTBE-, 91-01 UG-, and PS-6 UG-exposed mice. Because the decrease in relative uterine weight is suggestive of hormonal modulation, we evaluated the effects of MTBE, 91-01 UG, and PS-6 UGin vivoon hepatic 17-β estradiol metabolismin vitro.Gavage treatment with either blend of UG and with MTBE increased estrogen metabolism in isolated mouse hepatocytes. Hepatic microsomal P450 activity was assessed by 7-pentoxyresorufin-O-dealkylase (PROD) and 7-ethoxyresorufin-O-de-ethylase (EROD) activities. Similar increases in P450 content and PROD and EROD activities were observed in all exposed mice as compared to controls. No hepatoxicity was observed in any treatment group. The hepatic labeling index, as measured by the incorporation of 5-bromo-2′-deoxyuridine, was increased in all exposed mice at 3 days but not 21 days, indicating that MTBE and 91-01 UG are also hepatic mitogens. These data demonstrate that a newer blend of UG and the UG additive MTBE elicit short-term effects similar to those of PS-6 UG. Given that these effects are potentially related to tumor promotion and the general lack of genotoxic activity, MTBE and 91-01 UG may exhibit tumor promoting activity similar to that seen with PS-6 UG. Since the liver is under multihormonal control, the increase in hepatic estrogen metabolism and uterine effects support a potential role for endocrine modulations in both MTBE- and UG-induced hepatocarcinogenesis.

The influence of cytochrome P450 enzyme activity on the composition and quantity of volatile organics in expired breath

Abstract We have previously described a method to capture, identify and quantify volatile components in expired breath. The purpose of this research is to provide a non-invasive means to measure biomarkers of metabolism in vivo. In the present studies, the effect of 1-aminobenzotriazole (ABT), an inhibitor of diverse cytochrome P450 (P450) enzymes, on the composition of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with the catalytic activities and total content of hepatic P450. lntraperitoneal administration of ABT (100 mg kg-(1)) to rats resulted in markedly diminished hepatic microsomal P450 content and activities. The extent of inhibition was near maximal at 4 h, at which time approximately 50% of the total P450 content, about 65% of the CYPlA2 activity, 55% of the CYP2E1 activity, and about 80% of CYP2B activity were lost. Inhibition was maintained to 48 h post-dosing, but P450 content and activities had largely been restored by day 7. Concomitant with the inhibition of P450 were corresponding increases (up to several hundred-fold) in the molar amount of volatiles appearing in the breath of ABT-treated animals, and the rebound of P450 levels was attended by corresponding decreases in the appearance of breath volatiles. These studies indicate that P450 plays a major role in the metabolism of VOCs appearing in breath, and that these chemicals can serve as markers on P450 activity in vivo.