Phenotypic variability in induction of p-glycoprotein mrna by aromatic hydrocarbons in primary human hepatocytes

To determine whether human liver responds to treatment with aromatic hydrocarbons (AHs) with induction of the multidrug resistance (mdr) gene product P-glycoprotein and whether AH induction of mdr involves the Ah receptor, we compared induction of mdr mRNA with induction of cytochrome P450 (CYP)1A1 mRNA in AH-treated cultures of primary human hepatocytes. Hepatocytes from all 15 individuals tested responded to treatment with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with induction of CYP1A1 mRNA. However, only 62% and 55% of the preparations responded to treatment with MC and TCDD, respectively, with induction of mdr mRNA. Indeed, in some individuals mdr mRNA was suppressed by MC and TCDD despite robust CYP1A1 induction. These studies provide the first evidence that not only does individual variation in mdr induction by AH exist but that AHs regulate mdr in humans by a novel mechanism distinguishable from the classical Ah receptor pathway. The dramatic variability in AH induction of mdr may be a predictive risk factor that will help to identify an individual's risk of AH-associated toxicities. © 1995 Wiley-Liss Inc.     

Sixth aspen cancer conference: Molecular mechanisms of genetic deregulation in toxicity and carcinogenesis

The multidrug transporter P-glycoproteins are encoded by three multidrug-resistance (mdr) genes in rodents, designated mdr1a (mdr3), mdr1b (mdr1), and mdr2. Only the first two genes are functionally related to multidrug resistance. Activation of rodent mdr genes during liver regeneration and hepatocarcinogenesis has been reported. In mice, mdr1a is activated in hepatocellular carcinomas (HCCs) produced by various carcinogenic protocols, whereas both mdr1a and mdr2 are activated during liver regeneration. In this communication, we report isolating three gene-specific probes for the rat mdr homologues, which were used as probes in an RNase protection assay to demonstrate that mdr1b mRNA was expressed in HCCs induced by two different protocols. Furthermore, high levels of hepatic mdr1b mRNA but only moderate levels of mdr1a and mdr2 mRNA were seen in preneoplastic lesions in rats treated with 2-acetylaminofluorene. Likewise, highly elevated levels of hepatic mdr1b mRNA but only moderately increased levels of mdr1a and mdr2 mRNA were seen after partial hepatectomy. Nevertheless, the general patterns of tissue-specific expression of these three mdr genes were similar in rats and mice. These results reveal a complex hepatic gene expression pattern during hepatocarcinogenesis and hepatic proliferation for this conserved gene family in rodents.     

The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures

Primary cultures of rat hepatocytes were exposed to [ring-³H]-N-hydroxy-2-acetylaminofluorene(N-OH-AAF) for 4 h, and the RNA and DNA nucleoside adducts wereisolated and identified by h.p.l.c. The DNA adducts were shownto be N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF),N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxy-guanosin-8-yl)-2-acetylaminofluorene(dG-N2-AAF), while the RNA adducts were N-(guanosin-8-yl)-2-acetyl-aminofluorene,and N-(guanosin-8-yl)-2-aminofluorene. The removal of theseadducts was measured up to 38 h following the cessation of exposureof the hepatocytes to N-OH-AAF. The dG-C8-AAF adduct was removedwith a half-life of approximately 10 h, while dG-N²-AAF anddG-C8-AF remained constant for 14 h, followed by a slow rateof removal. The dG-C8-AAF adduct initially composed about 60%of the total DNA adducts of primary hepatocytes in contrastto the 20% found in liver in vivo. The formation of the 3 DNAadducts and the different rates of repair indicate that primarycultured rat hepatocytes may be a valuable system to study initiationof liver carcinogenesis by N-OH-AAF.     

Differential modulation of P-glycoprotein expression by dexamethasone and 3-methycholanthrene in rat hepatocyte primary cultures

Spontaneous and culture condition-dependent changes in P-glycoproteinexpression and activity have been monitored in primary culturesof rat hepatocytes by using immuno-blotting, PCR and fluorimetrictechniques. In hepatocytes cultured in basal medium withoutaddition of dexamethasone or 3-methylcholanthrene, mdr mRNAand P-glycoprotein increased progressively throughout a 72 hculture period, in concert with an enhancement in the abilityto extrude the fluorescent dye Rhodamine-123. Addition of 1µMdexamethasone to the culture medium slowed down the increasein mdr mRNA and P-glycoprotein, while inducing a significantincrease in the efficiency of R-123 efflux. Addition of either100 nM or 10 µM DEX produced different changes in mdrmRNA and protein, unrelated to the rate of Rhodamine-123 extrusion.When 50 µM 3-methylcholanthrene was added to the culturemedium in the absence of any hormone supplementation, no significantchanges in P-glycoprotein activity and expression took place,in comparison with control cultures. On the contrary, in thepresence of dexamethasone (100 nM and 1µM), 3-methylcholanthreneinduced an increase in mdr mRNA and in the amount of immunoblottableprotein during culture, without producing any concomitant increasein the efficiency to extrude Rhodamine-123. The last phenomenonresulted to be an artefact, since 3-methylcholanthrene was shownto inhibit Rhodamine-123 transport competitively. These resultsindicate that rat hepatocyte P-glycoprotein may be variouslymodulated in vitro, by supplementing culture medium with hormonesand/or xeno-biotics. Functional activity of the P-glycoproteinis not necessarily related with protein amount and/or mdr RNA.     

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity

DNA binding levels were determined and compared in culturedhepatocytes from male and female rats as well as other animalspecies following exposure to aflatoxin B₁ (AFB₁) or 2-acetylaminofluorene(2-AAF). When human, rat (both male and female) and mouse hepatocytesin primary culture were exposed to 2.0 ? 10^–7 M [³H]AFB₁(sp. act. 2.63 µCi/nmol) for 24 h, male rat hepatocyteshad the highest degree of [³H]AFB₁-DNA binding (203 pmol/mgDNA) and human hepatocytes contained the next highest bindinglevel (42 pmol/mg DNA). Hepatocytes from female rats contained38 pmol/mg DNA while cultured mouse hepatocytes contained only1.4 pmol/mg DNA. When the same dose of [³H]AFB₁ was administeredto the cultured male rat hepatocytes at 24 h, 48 h, 72 h and1 week after seeding, and incubated for 24 h, the DNA bindinglevels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallelexperiments to the cultured male rat hepatocytes above, theAFB₁-DNA binding levels in the cultured female hepatocytes were42, 41, 37 and 34 pmol/mg DNA respectively. Human, male andfemale rat hepatocytes in primary culture were exposed to 5.2? 10^–5 M 2-acetyl-amino [9–¹⁴C]fluorene (sp. act.0.0094 µCi/nmol) for 24 h. It was determined that malerat hepatocytes contained the highest amount of radioactivelylabeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), femalerat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytescontained 0.29 nmol/mg DNA. Results from our in vitro hepatocyteculture system correlate well with in vivo animal studies dealingwith species and sex differences in DNA binding and carcinogenicsusceptability. This indicates that hepatocytes in vitro maintainmany of the biological properties necessary for carcinogen responsesimilar to liver cells in vivo. In addition, comparison of genotoxiceffect in cultured hepatocytes from animals as well as humansmay be useful in evaluating carcinogenic potential of xenobioticsin human liver.     

Induction of CYP1A and glutathione S-transferase activities by 2,3,7,8-tetrachlorodibenzo-p-dioxin in human hepatocyte cultures

Induction of CYP1A and glutathione S-transferase activitieswith 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studiedin human hepatocytes in primary culture to investigate the variabilityof inducibility and the potency of TCDD. Determining inductionof 7-ethoxyresorufin O-deethylase activity, preferentially catalyzedby CYP1A isozymes, we obtained concentration—responsediagrams in TCDD-treated hepatocyte cultures from transplantdonors and patients undergoing hepatic surgery. At a concentrationof 10^–10 M TCDD approximately half-maximal induction ofCYP1A was observed. Northern analysis of CYP1A gene expressionshowed a similar concentration — response relationship.In comparison with rat hepatocytes, human hepatocytes were about10-fold less sensitive towards the CYP1A-inducing effect ofTCDD. No pronounced interindividual differences in the inducingpotency of TCDD (concentration which leads to half-maximal induction)were obvious in the six human individuals studied, whereas theefficacy of CYP1A induction was highly variable. In addition,inducibility of glutathione S-transferase (GST) activity alsorevealed a considerable degree of interindividual variation,i.e. a complete lack of induction in three out of six hepatocytepreparations and a highly variable efficacy of GST inductionamong responders which was not related to CYP1A inducibility.     

The 32P-post-labeling method in quantitative DNA adduct dosimetry of 2-acetylaminofluorene-induced mutagenicity in Chinese hamster ovary cells and Salmonella typhimurium TA1538

The usefulness of the ³²P-post-labeling/t.l.c. method for quantitativeDNA adduct dosimetry was evaluated. 2-Acetylaminofluorene (2-AAF)-DNAadducts from three systems were characterized qualitativelyand quantitatively by the ³H-radiolabeled technique with subsequentanalysis by h.p.l.c. (pre-labeling method) and by the ³²-post-labelingmethod. Both methods showed N-acetoxyacetylaminofluorene (N-OAc-AAF)reaction products with calf thymus DNA were predominantly N-(deoxyguanosm-8-yl)-2-acetylaminofhiorene(dG-C8-AAF) with some N-(deoxyguanosin-8-yl)-2-amino-fluorene(dG-C8-AF) and N-(deoxyguanosin-N²-yl)-2-acetyl-aminofluorene(dG-N2-AAF). In contrast, Chinese hamster ovary (CHO) cellstreated with [³H]N-OAc-AAF gave 80 or 90% dG-C8-AF adducts and20 or 10% dG-C8-AAF adducts with the post- or pre-labeling method,respectively. Likewise in CHO cells treated with 2-AAF in thepresence of rat liver homogenate, {small tilde}90% dG-C8-AFand 10% dG-C8-AAF adducts were detected using the ³²P-post-labelingmethod. In Salmonella typhimurium strain TA1538 treated with2-AAF or [³H]2-AAF in the presence of a rat liver homogenate,one adduct, dG-C8-AF, was identified. Similar quantitative resultswere also obtained with the two methods. However, the ³²P-post-labelingmethod was more sensitive and also eliminated the use of radiolabeled-mutagentreatments. Quantitative DNA adduct dosimetry was applied toAAF-induced mutagenesis in the S. typhimurium and CHO/HPRT mutationassays. A linear and reproducible relationship existed betweendG-C8-AF levels and AAF-induced mutants in both systems.     

Multiple cytochrome P-450 subfamilies are co-induced with P-glycoprotein by both phenothiazine and 2-acetylaminofluorene in rats.

We studied the effects of two P-glycoprotein (P-gp) inducers, 2-acetylaminofluorene (2-AAF) and phenothiazine (PTZ), administered intraperitoneally, on the activities and content of hepatic cytochrome P-450 (CYP) subfamilies in hepatic microsomes of Sprague-Dawley rats. After 4-day administration of 2-AAF or PTZ, the P-gp content was increased. The total CYP content after PTZ treatment was significantly increased compared with that of controls. The CYP1A, CYP2B and CYP3A2 contents were induced, while the CYP2C6, CYP2C11 and CYP2E1 contents remained unaffected. A marked increase in CYP1A1 was found after administration of each compound. Ethoxyresorufin O-deethylase, pentoxyresorufin O-deethylase, and testosterone 6beta hydroxylation activities showed a significant increase after both 2-AAF and PTZ treatments. In particular, ethoxyresorufin O-deethylase exhibited more than ten times greater activity than that of the controls after the treatments. These results suggest that P-gp inducers affect several CYP subfamilies in addition to CYP3A, which is reported to be up-regulated coordinately with P-gp by a CYP3A inducer.     

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene(N-OAc-AAF) have previously been shown to induce dose-dependentDNA strand breaks in primary hepatocytes from mice and rats.In an attempt to determine the relationship between the extentof DNA strand breaks and the formation of specific DNA-carcinogenbound adducts in murine liver, the capability of N-OH-AAF andN-OAc-AAF to induce both DNA single strand breaks and adductformation in in vivo and in primary hepatocytes was measured.N-OH-AAF induced a low level of DNA damage in F344 rats (10mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment.The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene(Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene(Gua-C8-AF) 34% versus 67% and Mguanin-N²-yl)-2-acetylaminofluorene(Gua-N²-AAF) 11% versus 10%, respectively, for rat and mouseliver. An additional unknown adduct (12%) was detected in mouseliver. Dose dependent DNA binding and formation of individualDNA adducts were observed in rat and mouse primary hepatocytesfollowing 1 h exposure to [ring-³H]-N-OH-AAF (0.1-20 µM)and [ring-³-N-OAc-AAF (5–20 /M). The patterns of DNA adductsin mouse and rat primary hepatocytes exposed to N-OH-AAF andN-OAc-AF were similar to those obtained in liver following invivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon(10^–4M) completely inhibited DNA damage induced by N-OH-AAFin mouse and partially in rat hepatocytes while DNA damage causedby N-OAc-AAF was only partially inhibited by paraoxon (10^–4M) in both species. Parallel experiments showed that paraoxon,at low concentration (10^– M), did not alter either thelevel of DNA binding or the pattern of adduct formation in rathepatocytes treated with N-OH-AAF (20 µM). However, at10^–4 M paraoxon partially blocked DNA binding (60%) andthe formation of Gua-C8-AAF (95%) and Gua-N²-AAF (80%) whileGua-C8-AF was increased twofold. In mouse hepatocytes paraoxonpretreatment (10^–4M) inhibited the formation of Gua-C8-AFby 70% following exposure to N-OH-AAF (20 µM). Gua-C8-AAFand Gua-N²-AAF were also inhibited but only at 10^–4M paraoxon.Paraoxon (10^–6 and 10^–4 M) pretreatment induceddosedependent partial inhibition of the covalent binding ofN-OAc-AAF to rat DNA and the formation of all guanine adducts.In the mouse, paraoxon (10^–6 and 10^–4 M) inhibitedthe formation of Gua-C8-AF while it increased Gua-C8-AAF. Theseresults indicate that a positive correlation exists betweenthe extent of DNA strand breaks and the formation of eitherGua-C8-AAF or Gua-C8-AF.     

A precursor--product relationship exists between oval cells and hepatocytes in rat liver

Oval cell proliferation was induced in twelve male Fischer ratsby administration of 2-acetylaminofluorene (2-AAF) for 2 weeksand by performing partial hepatectomy one week after the beginningof 2-AAF administration. Albumin expression in liver was studiedby using in situ hybridization of ³H-labeled rat albumin riboprobe.Radiolabeled thy-midine was administered to a group of animalsat day 6 after partial hepatectomy. The animals were killedat 0, 3, 7, 9, 11 and 13 days after partial hepatectomy. Bothoval cells and basophilk hepatocytes showed a prominent expressionof albumin, whereas albumin expression hi acidophilic preexistinghepatocytes was decreased. Oval cell nuclei were exclusivelylabeled one day after administration of [³H]thymidine. At day9, 11 and 13 basophilic hepatocytes became labeled and the areaoccupied by these cells increased. This is the first demonstrationof the transfer of radiolabeled thymidine from oval ceDs tonewly formed hepatocytes in vivo. Thus the precursor—productrelationship between oval cells and basophilic hepatocytes hasbeen established.     

Induction of human MDR1 gene expression by 2-acetylaminofluorene is mediated by effectors of the phosphoinositide 3-kinase pathway that activate NF-kappaB signaling

The expression of P-glycoprotein encoded by the multidrug resistance (MDR1) gene is associated with the emergence of the MDR phenotype in cancer cells. Human MDR1 and its rodent homolog mdr1a and mdr1b are frequently overexpressed in liver cancers. However, the underlying mechanisms are largely unknown. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression in cultured cells and in Fisher 344 rats. We recently reported that activation of rat mdr1b in cultured cells by 2-AAF involves a cis-activating element containing a NF-kappaB binding site located -167 to -158 of the rat mdr1b promoter. 2-AAF activates IkappaB kinase (IKK), resulting in degradation of IkappaBbeta and activation of NF-kappaB. In this study, we report that 2-AAF could also activate the human MDR1 gene in human hepatoma and embryonic fibroblast 293 cells. Induction of MDR1 by AAF was mediated by DNA sequence located at -6092 which contains a NF-kappaB binding site. Treating hepatoma cells with 2-AAF activated phosphoinositide 3-kinase (PI3K) and its downstream effectors Rac1, and NAD(P)H oxidase. Transient transfection assays demonstrated that constitutively activated PI3K and Rac1 enhanced the activation of the MDR1 promoter by 2-AAF. Treatment of hepatoma cells with 2-AAF also activated another PI3K downstream effector Akt. Transfection of recombinant encoding a dominant activated Akt also enhanced the activation of MDR1 promoter activation by 2-AAF. These results demonstrated that 2-AAF up-regulates MDR1 expression is mediated by the multiple effectors of the PI3K signaling pathway.     

Cytochrome P450 species involved in the metabolism of quinoline

Quinoline is a hepatocarcinogen in rats and mice and a well-knownmutagen in bacteria after incubation with rat liver microsomes.The specific cytochrome P450 enzymes involved in quinoline metabolismin human and rat liver microsomes were determined using cDNA-expressedcytochrome P450s, correlations with specific cytochrome P450-linkedmonooxygenase activities in human liver microsomes and inhibitionby specific inhibitors and antibodies. CYP2A6 is the principalcytochrome P450 involved in the formation of quinoline-1-oxidein human liver micro-somes (correlation coefficient r = 0.95),but is formed in only minute quantities in rat liver microsomes.CYP2E1 is the principal cytochrome P450 involved in the formationof 3-hydroxyquinoline (r = 0.93) in human liver microsomes andis involved in the formation in rat liver microsomes. A highcorrelation coefficient (r = 0.91) between CYP2A6 activity andquinoline-5,6-diol formation in human liver microsomes was observed,but this most likely reflects the involvement of CYP2A6 in theformation of quinoline-5,6-epoxide, from which the quinoline-5,6-diolis formed, as conversion of quinoline-5,6-epoxide to quinoline-5,6-diolon incubation of the epoxide with CYP2A6 could not be demonstrated.A cDNA-expressed human microsomal epoxide hydrolase, however,efficiently converted the epoxide to the diol and the microsomalepoxide inhibitor cyclohexene oxide inhibited quinoline-5,6-diolformation in rat liver microsomes. A preliminary kinetic analysisof quinoline metabolism in human liver microsomes was carriedout and Eadie-Hofstee plots indicate that the formation of quinoline-5,6-diolis monophasic, while that of quinoline-1-oxide and 3-hydroxyquinolineis biphasic.     

Role of cytochrome P450 in DNA damage induced by N-nitrosodialkylamines in cultured rat hepatocytes

The involvement of cytochrome P450 in the cytotoxicity and DNAdamage-repair induced by N-nitrosodipropylamine (NDPA), N-nitroso-n-butyl-n-propylamine(NBPA) and N-nitrosodibutylamine (NDBA) was investigated incultured hepatocytes isolated from untreated, phenobarbital(PB)-and pyridine (PYR)-pretreated rats. Pretreatment of ratswith PB caused a 10-fold increase in the sensitivity of hepatocytesto the cytotoxic actions of NDPA, NBPA and NDBA as measuredby trypan blue exclusion, whereas PYR pretreatment increasedthe sensitivity of hepatocytes to NDPA and NBPA, but not toNDBA. This elevated sensitivity correlated well with increased7-pentoxyresorufin depentylase activity catalyzed by P450 2B1in cultures from PB-pretreated rats and enhanced p-nitrophenolhydroxylase activity of P450 2E1 in cultures of hepatocytesfrom PYR-pretreated rats. Unscheduled DNA synthesis showed thatDNA damage-repair was significantly increased in freshly isolatedhepatocytes from PB- and PYR-pretreated rats. With increasingtime in culture, however, there was a marked reduction in theDNA damage repair response, concomitant with a decrease in thecytotoxicity of NDPA, NBPA and NDBA in primary cultures of hepatocytes.Coincident with this, a rapid loss in the specific activitiesof P450 2B1 and 2E1 was detected during the first 48 h in allprimary cultures. Although N-nitrosodimethylamine (NDMA), usedas a positive control, produced high nuclear grain counts incultures from PYR-pretreated rats, the toxic effect of NDMAin rat hepatocytes was much weaker than that observed with NDPA,NBPA and NDBA. This result suggests that the type of DNA damageor repair efficiency induced by NDPA, NBPA or NDBA might differfrom that due to NDMA.     

Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals

The broad spectrum of biological responses associated with exposureto 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is believedto be due to the alteration in expression of TCDD-induciblegenes. The aim of this study was to investigate the effectsof TCDD on the in vivo tissue-specific expression of the recentlyidentified TCDD-inducible cytochrome P450 CYP1B1 [Sutter etal. (1994) J. Biol. Chem., 269, 13092–13099] in Sprague-Dawleyrats. We cloned the 5.0 kb rat homolog of CYP1B1 from a TCDD-treatedrat liver cDNA library and showed that the rat and human CYP1B1predicted amino acid sequences are 80% identical. RNA hybridizationanalysis snowed that CYP1B1 is constitutively expressed in theadrenal glands and also in the testes of untreated rats. Thistissue distribution suggests that CYP1B1 may be a physiologicalsteroid hydroxylase. Seventy-two hours post-administration of25 µg/kg body wt TCDD by gavage, steady-state levels ofthe 5.1 kb CYP1B1 RNA were increased > 50-fold in liver,and to a lesser extent in kidneys, lung, heart and ovaries.Average CYP1B1 RNA levels were significantly higher in the kidneysand livers of TCDD-treated females than in those from similarlytreated males. In contrast, no significant sex-difference wasobserved in the levels of CYP1A1 in these tissues in TCDD-treatedanimals. In Sprague-Dawley rats, TCDD is a more potent hepatocarcinogenin females than in males. The induction of CYP1B1 in TCDD ratliver may be a contributing factor to the carcinogenic actionof this persistent environmental pollutant.     

Human hepatocyte-mediated mutagenesis and DNA repair activity

Combined cultures of human hepatocytes and human fibroblastsconstitute a system composed entirely of normal human cellsthat can be used to investigate the mutagenicity of chemicalsrequiring metabolic activation. Addition of diethylnilrosamine(DEN) to this system resulted in mutations at the hypoxanthine-guaninephosphoribosyltransferase locus of the human fibroblasts. Inseparate experiments with cultures of hepatocytes alone, DENinduced unscheduled DNA synthesis (UDS) in the human hepatocytes.A comparative analysis of UDS and hepatocyte-mediated mutagensisindicates a great degree of similarity between the human andpreviously studied rat hepatocytes in their response to DENin vitro.     

Lack of genotoxic activity of di(2-ethylhexyl)phthalate (DEHP) in rat and human hepatocytes

Di(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizerwhich has been reported to induce a statistically significantincrease in the incidence of hepatocellular carcinomas in femaleFischer-344 rats (8/50) when administered in the diet at 12000 p.p.m. for two years. Numerous studies with cells in culturehave failed to show any genotoxic activity associated with DEHP.Because DEHP induces multiple changes in the liver, such asperoxisomal proliferation, it was possible that these alterationscould result in genotoxic effects in the treated whole animalthat would not be seen in cells in culture. Accordingly, theability of DEHP to induce DNA damage or repair was examinedin rat hepatocytes in vivo and in vitro and in human hepatocytesin vitro. Unscheduled DNA synthesis was measured by incorporationof [³H]thy-midine into primary hepatocyte cultures immediatelyisolated from treated animals or hepatocyte cultures incubateddirectly with DEHP. DNA damage was measured by alkaline elutionof cellular DNA from the same cultures. In vivo-in vitro treatmentregimens were: (i) female rats, 12 000 p.p.m. DEHP in the dietfor 30 days; (ii) female rats, 12 000 p.p.m. in the diet for30 days, followed by 500 mg/kg DEHP by gavage 2 h before sacrifice;(iii) male rats, 500 mg/kg DEHP by gavage 2, 12, 24, or 48 hbefore sacrifice; and (iv) male rats, 150 mg/kg/day by gavagefor 14 days. In vitro conditions were 0.1, 1.0 and 10.0 mM DEHPin the cultures for 18 h. Primary cultures of human hepatocyteswere prepared from freshly discarded surgical material and exposedto the same concentration of DEHP. Concentrations up to 0.5mM mono(2-ethylhexyl)phthalate, a principal metabolite of DEHP,were also examined in the human hepatocyte assay. No chemicallyinduced DNA damage or repair was observed in vivo or in vitroin rat or human hepatocytes under any of the conditions employed.However, an increase in the percentage of cells in S-phase inthe animals given DEHP was observed. These data indicate thatDEHP does not exhibit direct genotoxic activity in the animalseven with a treatment regimen which eventually produced tumorsin a long term bioassay, and that both rat and human hepatocytesare similar in their lack of a genotoxic response to DEHP exposurein culture.     

Reactive oxygen species participate in mdr1b mRNA and P-glycoprotein overexpression in primary rat hepatocyte cultures

P-glycoproteins encoded by multidrug resistance type 1 (mdr1) genes mediate ATP-dependent efflux of numerous lipophilic xenobiotics, including several anticancer drugs, from cells. Overexpression of mdr1-type transporters in tumour cells contributes to a multidrug resistance phenotype. Several factors shown to induce mdr1 overexpression (UV irradiation, epidermal growth factor, tumour necrosis factor alpha, doxorubicin) have been associated with the generation of reactive oxygen species (ROS). In the present study, primary rat hepatocyte cultures that exhibit time-dependent overexpression of the mdr1b gene were used as a model system to investigate whether ROS might participate in the regulation of intrinsic mdr1b overexpression. Addition of H2O2 to the culture medium resulted in a significant increase in mdrlb mRNA and P-glycoprotein after 3 days of culture, with maximal (approximately 2-fold) induction being observed with 0.5-1 mM H2O2. Furthermore, H2O2 led to activation of poly(ADP-ribose) polymerase, a nuclear enzyme activated by DNA strand breaks, indicating that ROS reached the nuclear compartment. Thus, extracellularly applied H2O2 elicited intracellular effects. Treatment of rat hepatocytes with the catalase inhibitor 3-amino-1,2,4-triazole (2-4 mM for 72 h or 10 mM for 1 h following the hepatocyte attachment period) also led to an up-regulation of mdrlb mRNA and P-glycoprotein expression. Conversely, antioxidants (1 mM ascorbate, 10 mM mannitol, 2% dimethyl sulphoxide, 10 mM N-acetylcysteine) markedly suppressed intrinsic mdr1b mRNA and P-glycoprotein overexpression. Intracellular steady-state levels of the mdrl substrate rhodamine 123, determined as parameter of mdr1-type transport activity, indicated that mdr1-dependent efflux was increased in hepatocytes pretreated with H2O2 or aminotriazole and decreased in antioxidant-treated cells. The induction of mdr1b mRNA and of functionally active mdr1-type P-glycoproteins by elevation in intracellular ROS levels and the repression of intrinsic mdrlb mRNA and P-glycoprotein overexpression by antioxidant compounds support the conclusion that the expression of the mdr1b P-glycoprotein is regulated in a redox-sensitive manner.     

Cytochromes P450 in cynomolgus monkeys mutagenically activate 2-amino-3-methylimidazo(4, 5-f)quinoline (IQ) but not 2-amino-3, 8-dimethylimidazo(4, 5-f)quinoxaline(MeIQx)

THE PROMUTAGENIC AND PROCARCINOGENIC HETEROCYCLIC AMINES (HAS) FOUND IN COOKED MEATS ARE N-HYDROXYLATED BY MICRO-SOMAL CYTOCHROME P450 ENZYMES AS THE FIRST STEP IN THEIR METABOLIC ACTIVATION. IN CYNOMOLGUS MONKEYS, ONE OF THE HAS, 2-AMINO-3-METHYLIMIDAZO[4, 5-F: quinoline (IQ), has been shown to be a potent hepatocarcinogen.However, the structurally similar HA 2-amino-3, 8-dimethylimidazo[4,5-fquinoxaline (MelQx) lacks this potency to induce hepato-cellularcarcinoma in monkeys. Liver microsomes from cynomolgus monkeysshow a striking substrate specificity for the metabolic activationof IQ and MelQx, the former being a far better substrate forN-hydroxylation. Western blot analysis showed that cynomolgusmonkey hepatic microsomes constitutively express P450s immunologicallyrelated to the human CYP3A, CYP2C, and low levels of CYP1A1.For comparison, Western blot analysis of rat, human and patasmonkey microsomes was also carried out. Treatment of cynomolgusmonkeys with rifampicin induced hepatic cytochromes P450 relatedto human CYP3A4 and CYP2C9/10 without inducing CYP1A1 or CYP1A2.Immunoblot analysis also showed that chronic exposure of cynomolgusmonkeys to IQ induced hepatic microsomal cytochrome CYP1A1 andCYP1A2, similarly but lesser in magnitude to that observed with2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCCD) induction. Usingthe Ames Salmonella mutagenicity assay, we examined the effectof the inducers on the mutagenic activation (i.e. N-hydroxylation)of IQ and MelQx by cynomolgus monkey hepatic microsomes. Wealso examined the mutagenic activation of these HAs by rat,human and patas monkey liver microsomes. Microsomes from cynomolgusmonkeys treated with rifampicin showed a 3-fold increase inthe mutagenic activation of IQ but showed no increase in themutagenic activation of MelQx. Since cytochromes P4503A and/orP4502C are constitutively expressed in cynomolgus monkey hepaticmicrosomes, and upon induction with rifampicin are associatedwith an increased metabolic activation of IQ but not MelQx,it appears that CYP3A and/or CYP2C are the isoform(s) showingthe selective substrate specificity in the metabolic activationof IQ over MelQx. Treatment of monkeys with TCDD significantlyincreased the mutagenic activation of both IQ and MelQx, concomitantwith an induction of CYP1A isozymes. Thus, it appears that TCDD-inducibleCYP1A enzymes W-hydroxylate both substrates without selectivity.Together, these findings suggest that CYP3A and CYP2C are theprincipal isoforms in the cynomolgus monkey, associated withthe metabolic activation implicated in the induction of hepatocarcinogenicityby IQ. Furthermore, the poor metabolic activation of MelQx byCYP3A and CYP2C, coupled with low constitutive levels of CYP1Aisozymes, provide a metabolic explanation for the low hepatocarcinogenicpotency of MelQx in cynomolgus monkeys.