Low Cytochrome P450 Research Articles

Investigating the Correlation between Genotypes and Hepatic Protein Expression of CYP2C9, CYP2C19, CYP2D6, and CYP3A5 Using Postmortem Tissue from a Danish Population.

The cytochrome P450 (CYP) family of enzymes plays a central role in the metabolism of many drugs. CYP genes are highly polymorphic, which is known to affect protein levels, but for some low frequent CYP genotypes the correlation between genotype and CYP protein expression is less established. In this study, we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes of 250 Danish individuals included in a postmortem study. For 116 of the individuals, the hepatic CYP protein levels were investigated by a proteomics approach. Overall, we found the postmortem genetic and proteomic data to be in agreement with those of other studies performed on fresh hepatic tissue, showing the usability of postmortem hepatic tissue for this type of investigation. For less investigated genotypes, we could corroborate previously found results: 1) statistically significantly lower levels of hepatic CYP2C9 protein in individuals carrying the CYP2C9*3 variant compared with individuals with two wild type (wt) alleles; 2) comparable levels of CYP2C19 in CYP2C19*2/*17 and CYP2C19*1/*2 individuals; 3) reduced CYP2D6 protein levels in heterozygous individuals with the CYP2D6*3, CYP2D6*4, and CYP2D6*5 gene deletion variants; and 4) significantly lower levels of CYP3A5 protein in CYP3A5*3 homozygous individuals compared with individuals who were heterozygous for the CYP3A5*3 allele or homozygous individuals for the wt alleles. In conclusion, the use of postmortem tissue significantly increases the access to human specimens for research purposes, and postmortem proteomics can be used to investigate the link between CYP genotypes and hepatic protein expression. SIGNIFICANCE STATEMENT: In tissue samples from a large postmortem cohort (n = 250) we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes. Hepatic CYP protein levels were investigated in 116 individuals using a proteomics approach. For common genotypes, we found results similar to previous knowledge, pointing toward the usability of postmortem tissue. For the less investigated genotypes, we were able to corroborate genotype/protein expression correlations. It is a novel approach to use a large postmortem cohort to investigate genetic/protein expression correlations.

Effects of Chemicals in Reporter Gene Bioassays with Different Metabolic Activities Compared to Baseline Toxicity.

High-throughput cell-based bioassays are used for chemical screening and risk assessment. Chemical transformation processes caused by abiotic degradation or metabolization can reduce the chemical concentration or, in some cases, lead to the formation of more toxic transformation products. Unaccounted loss processes may falsify the bioassay results. Capturing the formation and effects of transformation products is important for relating the in vitro effects to in vivo. Reporter gene cell lines are believed to have low metabolic activity, but inducibility of cytochrome P450 (CYP) enzymes has been reported. Baseline toxicity is the minimal toxicity a chemical can have and is caused by the incorporation of the chemical into cell membranes. In the present study, we improved an existing baseline toxicity model based on a newly defined critical membrane burden derived from freely dissolved effect concentrations, which are directly related to the membrane concentration. Experimental effect concentrations of 94 chemicals in three bioassays (AREc32, ARE-bla and GR-bla) were compared with baseline toxicity by calculating the toxic ratio (TR). CYP activities of all cell lines were determined by using fluorescence-based assays. Only ARE-bla showed a low basal CYP activity and inducibility and AREc32 showed a low inducibility. Overall cytotoxicity was similar in all three assays despite the different metabolic activities indicating that chemical metabolism is not relevant for the cytotoxicity of the tested chemicals in these assays. Up to 28 chemicals showed specific cytotoxicity with TR > 10 in the bioassays, but baseline toxicity could explain the effects of the majority of the remaining chemicals. Seven chemicals showed TR < 0.1 indicating inaccurate physicochemical properties or experimental artifacts like chemical precipitation, volatilization, degradation, or other loss processes during the in vitro bioassay. The new baseline model can be used not only to identify specific cytotoxicity mechanisms but also to identify potential problems in the experimental performance or evaluation of the bioassay and thus improve the quality of the bioassay data.

Escaping from Flatland: Multiparameter Optimization Leads to the Discovery of Novel Tetrahydropyrido[4,3-d]pyrimidine Derivatives as Human Immunodeficiency Virus-1 Non-nucleoside Reverse Transcriptase Inhibitors with Superior Antiviral Activities against Non-nucleoside Reverse Transcriptase Inhibitor-Resistant Variants and Favorable Drug-like Profiles.

In the current landscape of antiretroviral options, there remains an urgent need for novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) with improved resistance profiles and safety properties. Herein, a series of novel tetrahydropyrido[4,3-d]pyrimidine derivatives were discovered utilizing the "escape from flatland" strategy. The most potent inhibitor 10c was endowed with broad-spectrum antiviral activity and improved resistance profiles against NNRTI-resistant variants compared to efavirenz and etravirine. Molecular simulations were investigated to furnish insights into the biological results. Drug-likeness assessment showed that 10c exhibited desirable physicochemical properties and in vitro metabolic stability. Notably, lower cytochrome P450 inhibition and human ether-à-go-go-related gene blockade liability were observed for 10c than those for etravirine and rilpivirine. Besides, 10c was characterized by excellent in vivo safety properties without acute/subacute toxicity and organ pathological damage. Overall, our multiparameter optimization campaign led to the identification of 10c with excellent antiviral activities and favorable drug-like profiles that could serve as an ideal drug candidate for further development.

Changes in Cytochrome P450 in Katahdin Ewes Fed Endophyte-infected Tall Fescue Seed Diets in Spring and Fall

Abstract Objective was to examine the activity of cytochrome P450 (CYP), a liver enzyme that metabolizes ergot alkaloids which is hindered by this fescue toxin, as an indicator of fescue toxicosis in sheep. In spring and fall, ewes were blocked by age and BW, and randomly assigned to 1 kg/ewe/d of endophyte-infected (E+; 61% of diet in fall, 13% of diet in spring; 0.8 µg/g of ergovaline; and soy hulls, alfalfa pellets, cottonseed hulls, molasses) or non-infected (E-; same proportion of E+/season and same feeds) tall fescue seed supplement (n = 10/diet in each season) for 28 d, plus hay, mineral and water. Ewes were exposed to teaser, d -21 to 0, and intact rams, d 0 (first day of diet) to 30. Rectal temperature (RT), serum concentration of prolactin (using RIA), and CYP were determined every 7–14 d between d 0–28, and pregnancy status on d 56. PromegaTM P450-Glo assay was used to determine CYP from serum. Data were analyzed by using PROC MIXED with repeated measures (SAS). Rectal temperature of E+ compared with E- ewes was elevated on at least one day in fall and spring (diet × season × d, P &amp;lt; 0.001). Serum prolactin was lower in E+ compared with E- ewes (diet × d, P &amp;lt; 0.001) and lower in fall (P &amp;lt; 0.001). CYP was higher in E- ewes on d 28 in fall (diet × season × d, P = 0.008), but otherwise similar between diets, and higher in fall than spring (P &amp;lt; 0.001). There was an unexpected negative correlation between CYP and prolactin (R = -0.24; P &amp;lt; 0.009), but there appears to be a subpopulation of ewes with low prolactin and lower CYP. CYP activity in sera may not be a marker for fescue toxicosis in ewes.

Discovery of piperidine-substituted thiazolo[5,4-d]pyrimidine derivatives as potent and orally bioavailable HIV-1 non-nucleoside reverse transcriptase inhibitors

HIV-1 reverse transcriptase offers a key target for antiviral therapy. However, the rapid emergence of drug-resistant mutations in reverse transcriptase as well as the poor pharmacokinetic properties of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) limits their clinical use. Starting from a previous piperidine-substituted thiophene[3,2-d]pyrimidine compound (K-5a2), here we explore the chemical space around the thiophene ring located in the solvent-exposed regions of the NNRTI binding pocket in detail. Bioisosterism-based structural modification leads to the discovery of a number of compounds as potent in vitro reverse transcriptase inhibitors, providing improved drug resistance profiles compared to the listed drug Etravirine. Furthermore, 14a and 19a are identified as lead compounds with good solubility, appropriate ligand efficiency, and lower cytochrome P450 liability. Compound 19a exhibits useful in vivo pharmacokinetic properties in rat and safety in mice, suggesting that it may have the potential to be an effective drug candidate for treating AIDS.

Metabolism of N-Methyl-2-Pyrrolidone in Honey Bee Adults and Larvae: Exploring Age Related Differences in Toxic Effects

In chronic feeding assays, the common agrochemical inert formulant N-methyl-2-pyrrolidone (NMP) is at least 20 times more toxic to honey bee larvae than to adults, but the underlying cause of this difference is unknown. In other taxa, NMP is primarily detoxified via a cytochrome P450 mediated pathway. Using a LC-MS method, putative cytochrome P450 metabolites of NMP were identified and quantified in adults and larvae following chronic exposure to NMP. Major differences in the identities and quantities of the generated metabolites were observed between adults and larvae. One major difference was the higher percentage of the administered NMP recovered as the parent compound in larvae compared to adults. To further explore the apparent difference in metabolic capacity, a spectrofluorometric method was used to compare general cytochrome P450 enzyme activity by monitoring the transformation of a 7-ethoxycoumarin substrate. Higher microsomal levels of 7-ethoxycoumarin-O-deethylase activity in adult fat bodies suggests that the higher percentage of unmetabolized NMP in larvae relative to adults may be due to lower cytochrome P450 enzyme activity in fat bodies. Taken together, these results suggest that larvae may be less able to detoxify xenobiotics encountered in diet than adults, and these findings will help inform future risk assessment.

Evaluation of the Interplay between Uptake Transport and CYP3A4 Induction in Micropatterned Cocultured Hepatocytes.

Previously we assessed the inductive response of prototypical inducers in hepatocyte monocultures and the long-term coculture model HepatoPac using cryopreserved hepatocytes from the same donors. We noted that the rifampicin EC50 generated using the HepatoPac model corresponded better to the EC50 based on clinical data compared with data generated in the monoculture system. We postulated that there may be differences in the functioning of uptake transporters between the two systems that may have led to the EC50 difference. In this study, we characterized the functional activity of multiple uptake transporters in the two systems using cryopreserved hepatocytes from the same donors. Our data suggest that uptake transporter activity is higher in HepatoPac compared with the monoculture system. As a follow up to this study, we measured the intracellular concentrations of rifampicin and bosentan, which are known substrates of uptake transporters; we observed significantly higher intracellular concentrations of both compounds in HepatoPac relative to the monoculture system. This finding equated to lower cytochrome P450 isoform 3A4 (CYP3A4) EC50 values in the HepatoPac system compared with the monoculture system for both mRNA and activity. In parallel, no significant EC50 shift was observed for carbamazepine and phenytoin, which are not known to be substrates of uptake transporters. Our data suggest that next generation liver models such as HepatoPac may be a useful in vitro tool to quantitatively predict drug-drug interactions when it is known that the perpetrator is also a substrate of drug transporters.

Development of an optimized cytotoxicity assay system for CYP3A4-mediated metabolic activation via modified piggyBac transposition

Drug-induced hepatotoxicity is often caused by cytochrome P450 (CYP)-dependent metabolism of drugs into reactive metabolites. Assessment of hepatotoxicity induced by bioactive compounds is hampered by low CYP expression within in vitro cell lines. To overcome this limitation, piggyBac transposition and monoclonal expansion were used to generate a HepG2 cell line with stable and homogenously high expression of CYP3A4, a prominent CYP isoform. Our studies demonstrate the generated line's constant CYP3A4 expression and activity for over 40 cell passages; to date, it has been in subculture for more than a year without addition of Puromycin. This cell line was utilized to evaluate cytotoxicity of two bioactive (troglitazone and acetaminophen) and two non-bioactive (citrate and galactosamine) compounds by MTT assay. Cell viability significantly decreased upon treatment with bioactive drugs. Moreover, cell lines used in the present study were more sensitive to toxic effects of troglitazone than previously reported. Therefore, this HepG2 cell-based assay system may provide a suitable hepatic model for predicting CYP3A4-mediated hepatotoxicity during preclinical drug development.

Protective effects of maslinic acid against alcohol-induced acute liver injury in mice

Protective effects of maslinic acid (MA) at 10, 15 or 20 mg/kg body weight/day against alcohol-induced acute hepatotoxicity in mice were examined. Mice were administrated by MA for 3 weeks, and followed by alcohol treatment. Results showed that MA pre-intake at three doses resulted in its accumulation in the liver; and dose-dependently lowered cytochrome P450 2E1 activity and protein expression at 23.5–51.2% and 21.4–62.3%, respectively (P < 0.05). MA pre-intake decreased subsequent alcohol-induced reactive oxygen species, interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, nitric oxide and prostaglandin E2 production; retained glutathione content; maintained catalase and glutathione peroxidase activities; and declined cyclooxygenase-2 and total nitric oxide synthase activities in the liver (P < 0.05). Furthermore, MA pre-intake suppressed 17.3–51.7% nuclear factor kappa (NF-κ)B p50, 23.5–58.8% NF-κB p65, 25.6–62.4% p-p38 and 24.1–63.0% p-JNK expression in the liver (P < 0.05). Histological data indicated that MA intake at test doses attenuated hepatic inflammatory infiltrate. These findings support that maslinic acid is a potent preventive agent against acute alcoholic liver disease.

The Effect of Aging on Acetaminophen Pharmacokinetics, Toxicity and Nrf2 in Fischer 344 Rats

We investigated the effect of aging on hepatic pharmacokinetics and the degree of hepatotoxicity following a toxic dose of acetaminophen. Young and old male Fischer 344 rats were treated with 800 mg/kg acetaminophen (young n = 8, old n = 5) or saline (young n = 9, old n = 9). Serum measurements showed old rats treated with acetaminophen had significantly lower serum alanine aminotransferase and higher acetaminophen and acetaminophen glucuronide levels and creatinine, compared with acetaminophen treated young rats (p < .05). Immunoblotting and activity assays showed old saline-treated rats had twofold lower cytochrome P450 2E1 activity and threefold higher NAD(P)H quinone oxireductase 1 protein expression and activity than young saline-treated rats (p < .05), although Nrf2, glutathione cysteine ligase-modulatory subunit, glutathione cysteine ligase-catalytic subunit, and cytochrome P450 2E1 protein expressions were unchanged. Primary hepatocytes isolated from young rats treated with 10 mM acetaminophen had lower survival than those from old rats (52.4% ± 5.8%, young; 83.6% ± 1.7%, old, p < .05). The pharmacokinetic changes described may decrease susceptibility to acetaminophen-induced hepatotoxicity but may increase risk of nephrotoxicity in old age.

Hepatobiliary Quiz-6 (2013)

1.Regarding anti-tubercular therapy (ATT) related hepatotoxicity the following are TRUE:1.Transient asymptomatic rise of transaminases is common and settles spontaneously with continuation of isoniazid.2.Most common pattern of ATT induced hepatotoxicity is cholestatic.3.ATT induced acute liver failure has worse prognosis virus related acute liver failure.4.Combination therapy increases the risk of hepatotoxicity.5.Daily dosing regimen increases risk of hepatotoxicity.2.Regarding the mechanisms of ATT related hepatotoxicity the following are TRUE:1.Slow acetylators have less risk of isoniazid induced hepatotoxicity.2.Rifampicin increases isoniazid induced hepatotoxicity by inducing cytochrome P450 enzymes.3.Rifampicin can cause both conjugated and unconjugated hyperbilirubinemia without hepatocellular damage.4.Pyrazinamide induced hepatotoxicity is dose dependent.5.Women have lower risk of ATT induced hepatotoxicity due to lower cytochrome P450 enzyme activity.3.Regarding model for end stage liver disease (MELD) the following are TRUE EXCEPT:1.MELD was initially developed as a score to predict survival following surgical porto-systemic shunting for refractory ascites or variceal bleeding.2.Upper value of serum creatinine is capped at 4 mg/dl.3.Addition of etiology of liver disease improves the predictive ability of the MELD score.4.MELD scores are also predictive of post transplant morbidity, length of ICU stay and cost of transplantation.5.Addition of other recipient and donor criteria to MELD may be useful in optimizing transplant risk in patients with high MELD scores.4.Regarding use of MELD for applications other than liver transplantation the following are TRUE EXCEPT:1.MELD score does not predict occurrence of infection and mortality in hospitalized patients with cirrhosis.2.MELD score can be used to estimate mortality after surgery in patients with cirrhosis.3.MELD score can be used to estimate hepatic failure after hepatic resection for hepatocellular carcinoma.4.MELD score does not predict mortality after hepatic resection for hepatocellular carcinoma.5.MELD is a predictor of rebleeding and mortality after variceal hemorrhage.5.Regarding development of non-alcoholic fatty liver disease (NAFLD) after liver transplant the following are TRUE:1.Recurrence of NAFLD in patients transplanted for cryptogenic cirrhosis is almost universal.2.De-novo development of NAFLD in patients transplanted for other indications is rare.3.NAFLD post liver transplant is an insidious process requiring months to years to develop.4.Risk factors for recurrent or de-novo NAFLD after transplant are similar to those for NAFLD in native liver.5.Well defined evidence based paradigms are available for treatment of NAFLD after liver transplant.6.Regarding hepatitis C virus (HCV) infection in patients of end stage renal disease (ESRD) on regular hemodialysis the following are TRUE:1.The prevalence of HCV infection is higher in ESRD patients than in the general population.2.Duration of hemodialysis is not a risk factor for HCV positivity.3.Guidelines routinely recommend use dedicated dialysis machines, patient isolation, and a ban on reuse of dialyzer in patients with HCV infection.4.Routine screening for HCV infection should be done in all ESRD patients on hemodialysis.5.HCV positive patients have worse outcome after renal transplant.7.Regarding gastric varices in patients with portal hypertension the following are TRUE EXCEPT:1.Gastric varices account for only 5–10% of all upper digestive bleeds.2.Bleeding from gastric varices is correlated with hepatic venous pressure gradient.3.Patients with extrahepatic portal vein obstruction are more likely to have isolated gastric varices than patients with cirrhosis.4.Transjugular intrahepatic porto-systemic shunt is the first line treatment of gastric varices.5.Balloon occluded retrograde transvenous obliteration is a newer modality for treatment of gastric varices.8.Regarding the mechanisms responsible for progression of steatosis to steatohepatitis in NAFLD the following are TRUE EXCEPT:1.Hepatic triglyceride is directly toxic to the liver and degree of steatosis is correlated with inflammation.2.Free fatty acids cause liver injury directly as well as via lipid peroxidation.3.Patients with steatohepatitis have higher oxidative stress compared with chronic viral hepatitis.4.Levels of inflammatory cytokines are similar in steatohepatitis and simple steatosis.5.Iron overload and hemochromatosis gene mutations are important cause of oxidative stress in Asian patients with steatohepatitis.9.Regarding primary cholestatic liver diseases the following are TRUE:1.Specific anti-nuclear antibodies directed against nuclear body or envelope proteins are useful for diagnosis of primary biliary cirrhosis (PBC).2.The Barcelona and Paris criteria are used to asses response to therapy in primary sclerosing cholangitis (PSC).3.Ursodeoxycholic acid decreases risk of colonic neoplasia in PSC.4.Colectomy prior to liver transplantation decreases risk of recurrence of PSC after transplantation.5.Immunoglobulin G4-associated cholangitis is steroid unresponsive.10.Regarding evaluation of a patient with ascites the following are TRUE EXCEPT:1.Bedside inoculation of ascitic fluid into blood culture bottles increases bacterial detection.2.Yield of malignant cytology improves if multiple samples of fluid are tested.3.CA 125 is a useful investigation in a patient with suspected malignant ascites.4.Serum-ascites albumin gradient (SAAG) ≥ 1.1 is has good accuracy for underlying portal hypertensive ascites even in cases of mixed ascites.5.Pro-brain natriuretic peptide cannot differentiate ascites due to heart failure from ascites due to cirrhosis.

Antagonism of tamoxifen and antidepressants among women with breast cancer

Tamoxifen is used as an adjuvant therapy to reduce breast cancer recurrence among women with estrogen receptor positive tumors. Antidepressants are also commonly used in such women, to treat depression or to manage hot flushes, a frequent tamoxifen secondary effect. Some antidepressants could potentially inhibit cytochrome P450 2D6, required to activate tamoxifen, interfering with its action. Although there is not a clear cut directive on the subject, it is nowadays recommended to treat women with antidepressants with the lower cytochrome P450 2D6 inhibition potential to avoid a possible antagonism that may reduce tamoxifen s prevention of breast cancer recurrence at least in some patients with CYP2D6 genetic variation. The recommended antidepressants are desvenlafaxine, milnacipran, venlafaxin, escitalopram and citalopram.

Tamoxifeno y antidepresivos: ¿Antagonistas en la prevención del cáncer de mama?

Tamoxifen is used as an adjuvant therapy to reduce breast cancer recurrence among women with estrogen receptor positive tumors. Antidepressants are also commonly used in such women, to treat depression or to manage hot flushes, a frequent tamoxifen secondary effect. Some antidepressants could potentially inhibit cytochrome P450 2D6, required to activate tamoxifen, interfering with its action. Although there is not a clear cut directive on the subject, it is nowadays recommended to treat women with antidepressants with the lower cytochrome P450 2D6 inhibition potential to avoid a possible antagonism that may reduce tamoxifen s prevention of breast cancer recurrence at least in some patients with CYP2D6 genetic variation. The recommended antidepressants are desvenlafaxine, milnacipran, venlafaxin, escitalopram and citalopram.

Comprehensive characterization of cytochrome P450 isozyme selectivity across chemical libraries

The cytochrome P450 (CYP) gene family strongly influences drug development. We determined potency values for 17,143 compounds against recombinant CYP 1A2, 2C9, 2C19, 2D6, and 3A4 enzymes through an in vitro bioluminescent assay. The compound collections included substances from typical libraries and FDA-approved drugs. Cross-library isozyme inhibition (30–78%) was observed with important differences between collections. While only 7% of the typical screening library was inactive against all five isozymes, 33% of FDA-approved drugs were inactive, reflecting the optimized pharmacological properties of the latter. Unexpectedly, drugs exhibited less activity towards the CYP 2C9 and 2C19 isozymes compared to un-optimized collections. We then identified substructures that differentiated between the five isozymes as well as substructures trending towards active or inactive categories. We describe here a pharmacological compendium to further the understanding of CYP isozymes.

Biomarker Responses and Decreasing Contaminant Levels in Ringed Seals (Pusa hispida) from Svalbard, Norway

Blubber was analyzed for a wide range of contaminants from five subadult and eight adult male ringed seals sampled in 2004, namely, for polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), toxaphenes, chlordanes, dichlorodiphenyldichloroethylene (DDE), and polybrominated diphenylethers (PBDEs). Contaminant levels were compared to previously sampled animals from the same area, as well as data from literature for other arctic wildlife species from a wide variety of locations. Ringed seals sampled in 2004 showed 50–90% lower levels of legacy contaminants such as PCBs and chlorinated pesticides compared to animals sampled in 1996 of similar age (14 subadults and 7 adult males), indicating that the decline of chlorinated contaminants observed during the 1990s in a variety of arctic wildlife species is continuing into the 21st century. The results also indicated that PBDE declined in ringed seals; levels in 2004 were about 70–80% lower than in animals sampled in 1998. This is one of the first observations of reduced exposure to these compounds and might be a first indication that restrictions of production and use of these contaminants have resulted in lower exposures in the Arctic. The PCB pattern shifted toward the less chlorinated (i.e., less persistent) PCBs, especially in adult ringed seals, possibly as a result of reduced overall contaminant exposures and a consequently lower cytochrome P-450 (CYP) induction, which results in a slower metabolism of less persistent PCBs. The overall effect would be relative increases in the lower chlorinated PCBs and a relative decreases in the higher chlorinated PCB. Possibly due to low exposure and consequent low induction levels, ethoxyresorufin O-deethylation (EROD) activity proved to be a poor biomarker for contaminant exposure in ringed seals in the present study. The close negative correlation (r 2 = 70.9%)between EROD activity and percent blubber indicates that CYP might respond to increased bioavailability of the contaminant mixtures when they are mobilized from blubber during periods of reduced food intake.

Hepatic pharmacokinetics of propranolol in rats with adjuvant-induced systemic inflammation

Systemic inflammation is known to affect drug disposition in the liver. This study sought to relate and quantitate changes in hepatic pharmacokinetics of propranolol with changes in hepatic architecture and physiology in adjuvant-treated rats. Transmission electron microscopy was used to assess morphological changes in mitochondria and lysosomes of adjuvant-treated rat livers. The disposition of propranolol was assessed in the perfused rat liver using the multiple indicator dilution technique. Hepatic extraction and mean transit time were determined from outflow-concentration profiles using a nonparametric method. Kinetic parameters were derived from a two-phase physiologically based organ pharmacokinetic model. Possible relationships were then explored between the changes in hepatic drug disposition and cytochrome P-450 activity and iron concentration. Adjuvant treatment induced the appearance of mitochondrial inclusions/tubularization and irregularly shaped lysosomes in rat livers. Livers from adjuvant-treated rats had (relative to normal) significantly higher alpha(1)-acid glycoprotein (orosomucoid) and iron tissue concentrations but lower cytochrome P-450 content. The hepatic extraction, metabolism, and ion trapping of propranolol were significantly impaired in adjuvant-treated rats and could be correlated with altered iron store and cytochrome P-450 activity. It is concluded that adjuvant-induced systemic inflammation alters hepatocellular morphology and biochemistry and consequently influences hepatic disposition of propranolol.

Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells

1. Cultured hepatic cells have reduced cytochrome P450 (CYP) activities in comparison with human liver, but the mechanism(s) that underlies this circumstance is not clear. We investigated the causes of this low CYP activity by analysing the activity, protein, mRNA and heterologous nuclear RNA contents of the most important CYPs involved in drug metabolism (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) in cultured human hepatocytes, and in HepG2 and Mz-Hep-1 hepatoma cell lines. 2. After 24 h of culture, hepatocytes retained most of their CYP activities and protein contents, but the mRNA decreased 20-fold. However, the mRNA content of most CYPs in 24-h hepatocytes was still 400-fold higher than in hepatoma cells. When we examined the transcriptional activity of the CYP genes, this decreased during culture time in hepatocytes and it was poor in hepatoma cell lines. 3. We investigated the abundance of key hepatic transcription factors that govern CYP transcription (C/EBP- β : LAP and LIP, HNF-3 α, HNF-4 α, RXR- α) and observed that the expression of some factors was altered in the hepatoma cells. 4. In conclusion, the loss of biotransformation activity in cultured hepatic cells is caused by a decrease in CYP transcription, which correlates with an alteration in the expression of key transcription factors.

Morphology and cytochrome P450 isoforms expression in precision-cut rat liver slices

Precision-cut liver slices are a widely accepted in vitro system for the examination of drug metabolism, enzyme induction, or hepatotoxic effects of xenobiotics. The maintenance of the distinct lobular expression and induction pattern of phase I biotransformation enzymes, however, has not been examined systematically so far. Thus, in the present study, both longitudinal and transversal sections of male rat liver slices were investigated morphologically, as well as immunohistochemically for the expression of different cytochrome P450 (CYP) isoforms after prolonged incubation or after exposure to typical inducers. Histopathological examinations revealed an increasing vacuolization of the periportal hepatocytes mainly in the middle of the slices from 6 h of incubation on, paralleled by a loss of glycogen in the respective cells. After 24 h, mainly in the center of the slices, necroses of cells occurred. After 48 h of incubation, typically a central band of coagulative necrosis flanked by superficial layers of viable cells was observed. Freshly prepared slices displayed a CYP subtypes expression as normal liver specimen, a very low centrilobular CYP 1A1 immunostaining, but a strong CYP 2B1 and 3A2 expression predominantly in the central and intermediate lobular zones. From 2 h on, the immunostaining for CYP 2B1 and 3A2 was to some extent reduced. After 24 h of incubation with β-naphthoflavone, the CYP 1A1 and 2B1 expression was induced mainly in the viable cells around central veins, around some portal fields with bigger vessels and in the cell layers close to the slice surface. At the same sites, phenobarbital led to an increased CYP 2B1 and 3A2 expression and dexamethasone to an elevated CYP 3A2 immunostaining. These results show, that an in vitro induction of phase I enzymes in precision-cut liver slices can be demonstrated also immunohistochemically.

Prostaglandin H synthases and their importance in chemical toxicity.

The metabolism of a xenobiotic is an important stage resulting in its toxification (bioactivation) or detoxification. The most common reaction is the oxidation catalyzed by the cytochrome P450 (CYP) enzyme system. An alternate enzyme for chemical oxidation is the prostaglandin H synthase (PGHS) also known as cyclooxygenase (COX). The PGHS is the initial enzyme in arachidonate metabolism and formation of prostanoids such as prostaglandins (PG), prostacyclins, and thromboxanes. However, 25 years ago it was found that during the reduction of the endogenous substrate, hydroperoxy-endoperoxide (PGG2) to hydroxy-endoperoxide (PGH2), the PGHS enzyme is capable to "co-oxidize" chemicals. In this reaction, a broad spectrum of chemicals can serve as electron donors such as phenolic compounds, aromatic amines, and polycyclic aromatic hydrocarbons. In contrast to numerous CYP enzymes in liver, the PGI is an alternate enzyme for xenobiotic metabolism in extrahepatic tissues. In respect of tissue distribution, PGHS can play an essential role in the bioactivation of e.g. procarcinogenic chemicals in certain target tissues that possess low CYP monooxygenase activity. Two PGHS isozymes have been identified: PGHS-1 and PGHS-2, which have very similar kinetic properties, but differ in regard to expression and regulation. In recent studies it was shown that not only endogenous stimuli but also drugs and environmental chemicals can activate PGHS-2 expression. Therefore the PGHS enzymes provide two interesting aspects for pharmacology and toxicology: a) the co-oxidation of chemicals and b) the altered synthesis of prostanoids after exposure to certain xenobiotics which can be essential for their ultimate toxicity.

Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells.

The heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are pyrolysis products formed when meat is cooked and are rodent mammary carcinogens. They are thought to be metabolically activated by N-hydroxylation, catalysed by cytochrome P450 (CYP), followed by O-acetylation catalysed by N-acetyltransferases. Primary cultures of human mammary epithelial cells (HMECs) prepared from up to 26 individuals for each compound, were treated with IQ, MeIQ, or PhIP (500 microM) or with N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) or N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ) (20 microM) and the levels of adduct formation in their DNA analysed by 32P-post-labelling. In order to investigate whether pharmacogenetic polymorphisms influence DNA adduct formation, the NAT2 genotype of each individual was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that distinguishes between the wild-type and four variant alleles. Presence of two variant alleles designates a slow NAT2 acetylator, whereas individuals with one or two wild-type alleles are designated fast NAT2 acetylators. Interindividual variations in total DNA adduct levels ranged for IQ from 0.64-63.1 DNA adducts per 10(8) nucleotides (mean 7.80), for MeIQ from 1.99-17.8 (mean 6.63), for PhIP from 0.13-4.0 (mean 0.96), for N-OH-PhIP from 6.32-497 (mean 176) and for N-OH-IQ from 0.92-30.6 (mean 9.24). The higher adduct levels observed in cells treated with the N-OH metabolites suggests that N-hydroxylation is the rate-limiting step in HMECs and this may be due to low CYP levels. In contrast, the Phase II reaction catalysed by N-acetyltransferases is probably the major step in the metabolic activation of heterocyclic amines that occurs in the breast. Higher mean levels of heterocyclic amine-DNA adduct formation were detected in the cells of NAT2 fast acetylators compared with slow acetylators, with mean adduct levels per 10(8) nucleotides following IQ treatment, of 12.74 and 3.57 respectively, following PhIP treatment, of 1.20 and 0.74, respectively, following MeIQ treatment, of 7.90 and 5.08, respectively and following N-OH-PhIP-treatment, of 243.1 and 130.0, respectively. However, due to the large variations in adduct levels, these differences in mean values were not statistically significant with the limited number of individuals studied. This appears to be the first pilot study to demonstrate interindividual variations in the metabolic activation of heterocyclic amines and their metabolic intermediates in primary cultures of HMECs in vitro.