Species differences in the cytotoxic and genotoxic effects of 2-acetylaminofluorene and its primary metabolites 2-aminofluorene and N-OH-2-acetylaminofluorene

2-Acetylaminofluorene (AAF), 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene(N-OH-AAF) could be activated to mutagens in S. typhimuriumusing either 9000 g supernatant (S9) or hepatocytes isolatedfrom rats, mice, hamsters or guinea pigs. Their relative mutagenicpotency was generally N-OH-AAF > AF > AAF. Monolayer culturesof hepatocytes exposed to AAF/AF/N-OH-AAF showed evidence ofDNA damage measured as unscheduled DNA repair synthesis. Theorder of activity in rat and hamster was N-OH-AAF > AAF >AF, in guinea pig and mouse N-OH-AAF > AF > AAF. OnlyN-OH-AAF caused observable cytotoxicity, and the rat hepatocyteswere the far more sensitive species. Neither the resistanceof guinea pig liver nor the greater susceptibility of the ratliver to the carcinogenic effects of AAF and N-OH-AAF couldbe readily explained by the species differences in activatingthese compounds to mutagens in Salmonella or to DNA damagingagents in the hepatocytes. It is possible that cytotoxic effectsof N-OH-AAF may be of some importance for the observed speciesdifferences in the liver carcinogenic effects of AAF and N-OH-AAF.     

Metabolism and activation of 2-acetylaminofluorene in isolated rat hepatocytes.

The metabolism of 2-acetylaminofluorene (AAF) as well as the activation of AAF to covalently bound and mutagenic intermediates were studied in isolated rat hepatocytes. The cell system readily formed oxidized, deacetylated, and conjugated AAF metabolites. Pretreatments of animals with the inducer beta-naphthoflavone led to increases in phenolic and conjugated as well as covalently protein-bound products. Addition of 4-nitrophenol, a substrate for conjugation, increased the levels of free phenols and inhibited the formation of water-soluble metabolites. At the same time, the rates of covalent protein binding were decreased. Formation of 9-hydroxy-2-acetylaminofluorene could also be demonstrated. The pathway leading to this alicyclic hydroxylated AAF metabolite was not induced by prior beta-naphthoflavone treatment, nor was it inhibited by 4-nitrophenol addition. The cells converted AAF as well as aminofluorene and 2,4-diaminoanisole to mutagenic intermediates which were released into the incubation medium. 2-Aminofluorene was considerably more mutagenic than was AAF in this system. Addition of microsomes increased the mutagenicity of AAF, but not that of 2-aminofluorene or 2,4-diaminoanisole, presumably by deacetylation of N-hydroxy-2-acetylaminofluorene to N-hydroxy-2-aminofluorene.     

Mutagenicity and DNA damage induced by arylamines in the Salmonella/hepsdocyte system

Coincubation of isolated, intact rat hepatocytes with Salmonellatyphimurium tester strain TA 98 {Salmonella/ hepatocyte system)has been employed to determine both bacterial mutagenicity andDNA damage in rat hepatocytes following treatment with 2-acetylaminofluorene(AAF) and its derivatives. In vivo pretreatment of rats witheither 2,3,7,8-tetrachlorodibenzodioxin or 3-methylcholanthrenemarkedly increased both DNA damage and bacterial mutation frequencyupon incubation of AAF or 2-aminofluorene (AF) in this system.The increase in damage to the hepatocyte DNA was more pronouncedafter AAF treatment than following AF exposure, while the increasein bacterial mutation frequency was greater after AF treatmentTreatment of hepatocytes with paraoxon prior to exposure toN-hydroxy-2-acetylaminofhiorene (N-OH-AAF) or N-acetoxy-2-acetyl-aminofluorene(N-OAc-AAF) partially inhibited both DNA damage and the bacterialmutagenicity caused by these agents. Treatment of primary rathepatocytes with 2-hydroxy-2-aminofluorene (N-OH-AF) causesa low level of DNA breaks. Substitution of primary rat hepatocyteswith highly differentiated rat hepatoma cells (Reuber H4-II-E)revealed a low level of DNA breakage after exposure to N-OH-AAFwhereas treatment with either N-OAc-AAF or N-OH-AF induced adose dependent increase in DNA breaks. Pretreatment of the Reubercells with paraoxon inhibited the DNA damage caused by N-OAc-AAFwhereas the DNA damage induced by N-OH-AF was increased afterparaoxon treatment. Employing host cells with differing metaboliccapacity, such as Reuber vs. primary hepatocytes, in the Salmonella/hepatocytesystem, may allow a determination of the relative importanceof different metabolic pathways in mutagenicity and/or genotoxicityof arylamines.     

Metabolism of acetylaminofluorene in primary cultures of human hepatocytes: dose-response over a four-log range

The metabolism of acetylaminofluorene (AAF) in human hepatocytecultures from different donors was investigated for a four-logconcentration range (500, 50, 5.0 and 0.5 µM) or at 3,8 and 24 h at 500 µM. The metabolite profile was dependenton the concentration to which the cells were exposed. The hepatocytecultures varied in the degree to which they metabolized AAFpredominantly because of different levels of deacetylation.Ring-hydroxylation was the predominant pathway for AAF metabolismat low concentrations (5.0 and 0.5 µM) but saturated inthree of four human cases at high concentrations of AAF; N-hydroxylationdid not appear to become saturated. Human hepatocytes catalyzedthe covalent binding of AAF metabolites to their DNA. A linearincrease in DNA binding was observed when increasing concentrationsof AAF were added to hepatocyte cultures; however, the increasein AAF metabolites binding to DNA was not proportional to thedose. While the concentration of AAF in the media was increasedover a four-log range, both the production of N-hydroxy AAFand binding of metabolites to hepatocellular DNA increased overapproximately a three log range. These results with culturedhuman hepatocytes indicate that the pathways of AAF metabolismare qualitatively similar to those identified in experimentswith rat hepatocytes as well as experiments conducted in vivowith human subjects. These studies confirm that the culturedhuman hepatocyte is a useful model for the investigation ofhuman xenobiotic metabolism and indicate that the concentrationof the xenobiotic used in the experiments is an important determinantof the metabolitic profile produced.     

The genotoxicity of aromatic amines in primary hepatocytes isolated from C57BL/6 and DBA/2 mice

The capacity of the chemical carcinogen 2-acetylaminofluorene(AAF) and its derivatives to cause DNA damage in primary mousehepatocytes from aryl-hydrocarbon responsive C57BL/6 and non-responsiveDBA/2 mice was studied using the alkaline elution technique.Low levels of DNA damage were observed after exposure of hepatocytesto either AAF or 2-aminofluorene (AF) (50 –100 µM).Quantitation of metabolites produced from AAF in hepatocytesfrom untreated C57BL/6 and DBA/2 mice using h.p.l.c. showeda similar metabolic profile with respect to C- and N-hydroxylations.After in vivo pretreatment with the potent monooxygenase inducerTCDD (50 µg/kg), N-hydroxylation in the C57BL/6-and DBA/2-derivedhepatocytes increased 25- and 5-fold, respectively. However,the C-hydroxylation pathways were still responsible for 90%of the metabolism in cells from both strains. This may explainwhy only a slight increase in the DNA damage was observed inC57BL/6 mouse hepatocytes after incubation with AF or AAF andno increase in DNA damage was seen in the DBA/2 hepatocytesisolated from TCDD treated animals. Both N-hydroxy-2-acetyl-aminofluorene(N-OH-AAF) and N-acetoxy-2-acetylamino-fluorene (N-OAc-AAF)caused clear dose-dependent increases in DNA strand breaks (5–100 M), suggesting that N-hydroxylation was the ratelimiting step in the activation process of AAF leading to theDNA damage. Treatment of hepatocytes with paraoxon, an inhibitorof microsomal deacetylase activity, prior to exposure to eitherN-OH-AAF or N-OAc-AAF completely inhibited the damage causedby N-OH-AAF, while the damage caused by N-OAc-AAF was only partiallyinhibited. This suggests that these compounds are causing genotoxiceffects after deacetylation. In accordance with this, N-hydroxy-2-aminofluorene(N-OH-AF), the deacetylated metabolite of N-OH-AAF, was an effectivegenotoxic agent, causing DNA strand breaks at low doses. Depletionof cellular glutathione by pretreatment with diethyl maleate,increased the sensitivity of the cells to the damage inducedby N-OH-AF. These data indicate that glutathione may play animportant role in the detoxification of N-OH-AF in mouse hepatocytes.     

Metabolism of 2-acetylaminofluorene by two 3-methylcholanthrene-inducible forms of rat liver cytochrome P-450

The present study examines the contribution of two major 3-methylcholanthrene (3-MC)-inducible forms of rat liver cytochrome P-450 (P-448MC and P-448HCB) to the metabolism of 2-acetylaminofluorene (AAF). In a reconstituted enzyme system, purified rat liver P-448MC metabolized AAF at a 10-fold greater rate than P-448HCB. The major metabolites produced by cytochrome P-448MC were 3-hydroxy (OH) (30%), 5-OH (24%), 7-OH (22%), and 9-OH (10%). N-OH-AAF (3%) was a minor metabolite. In contrast, P-448HCB catalyzed the N-hydroxylation of AAF preferentially (15% of total metabolites). The other primary metabolites formed by this isozyme were 7-OH-AAF (30%), 5-OH-AAF (29%), and 9-OH-AAF (25%). Cytochrome P-448HCB catalyzed the formation of less 3-OH-AAF (7%) than did P-448MC (30%). Since cytochrome P-448HCB is immunochemically related to P-448MC, specific antisera to both isozymes were made by immunoabsorption with the appropriate antigen bound covalently to Sepharose. Anti-P-448MC inhibited AAF metabolism approximately 43% in microsomes from 3-MC-induced male rats and 30% in microsomes from rats treated with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), another 3-MC-type inducer. Anti-P-448HCB inhibited total metabolism of AAF by only 22 and 38% in microsomes from 3-MC- and HCB-induced rats. However, anti-P-448HCB inhibited N-hydroxylation by 60% in both 3-MC- and HCB-induced microsomes. Anti-P-448MC did not inhibit N-hydroxylation. Neither antibody affected AAF metabolism in control microsomes. These data suggest that, in rat liver, two 3-MC-inducible izozymes of cytochrome P-450 metabolize AAF; however, N-hydroxylation is catalyzed primarily by one of these isozymes, cytochrome P-448HCB.     

Bovine bladder and liver cell and homogenate-mediated mutagenesis of Salmonella typhimurium with aromatic amines

Comparisons of aromatic amine activation were made by usingintact cells or cell homogenates (S-9) from bovine bladder urotheliumand liver. Since both liver and bladder are thought to contributecarcinogenic metabolites for bladder cancer initiation, comparisonsof these two organs' relative ability to activate aromatic aminesto mutagens were made. Salmonella typhimurium mutagenesis wasused as an indication of mutagen production. Activation occurredin a dose dependent manner and no mutagenic response occurredunless activating cells or S-9 were present. Bladder urothelialcells metabolically activated the carcinogens, 2-amino-fluorene(AT), 2-acetylaminofluorene (AAF), 4-aminobi-phenyl (4ABP),benzidine (BZ), and 2-naphthyIamine (2NA) but not 1-naphthylamine(1NA), a non-carcinogen. Liver cells were less active than bladdercells in activating AF and AAF; but there was little or no hepatocyteactivation of 4ABP, BZ, 2NA and 1NA. Intact bladder cells weremore effective than bladder S-9 in activating AAF, but not inactivating AF, 4ABP, BZ and 2NA. The liver S-9 showed more mutagenicactivity with AF than did intact liver cells; the reverse wastrue for AAF, and bovine liver S-9 showed little or no activationof 4ABP, BZ, and 2NA. 1NA was not activated by either S-9 preparation.As was the case for intact cells, bladder S-9 was more effectivethan liver S-9 in activating the aromatic amines studied. Theresults demonstrate the capacity of bovine bladder urotheliumto metabolically activate aromatic amines and suggest a rolefor the target organ itself in carcinogen activation. Informationon the relative usefulness of intact cells versus S-9 preparationsas activation systems was also obtained from these studies.     

Consequences of 3-methylcholanthrene-type induction for the metabolism of 4-aminobiphenyl in isolated rat hepatocytes

Carcinogenic aromatic amines such as 4-aminobiphenyl (4-ABP)are extensively metabolized by both oxidative and conjugationreactions. Thus the burden of genotoxic metabolites of 4-ABPin a target organ is probably influenced by the balance of N-hydroxylationand alternative metabolic pathways in the hepatocyte. In freshlyisolated rat hepatocytes, 4-ABP (at a substrate concentrationof 10 µM) was mainly N-acetylated (54% of total metabolites),while 2% Ar-hydroxy-4-ABP-N-glucuronide and 21% of unconjugatedN-hydroxylated metabolites were detectable. Ring-hydroxylatedmetabolites and the primary N-glucuronide of 4-ABP accountedfor 8% and 4%, respectively. Pretreatment of rats with 3-methylcholanthrene(MC), a dioxin-type inducer of CYP1A isozymes and phenol UDP-glucuronosyltransferase(UGT1A1), led to a dramatic decrease of W-acetylated (2% oftotal metabolites) and an increase of N-hydroxylated (54% asfree and glucuronidated compound) and ring-hydroxylated (35%)metabolites. Essentially similar effects were seen at a substrateconcentration of 50 µM. Consistently, MC-type inductionwith ß-naphthoflavone resulted in a significant increasein the formation of DNA adducts of 4-ABP, detected by ³²P-postlabellingof hepatocellular DNA. The results suggest that, similar toa previous study with 2-naphthylamine (2-NA), MC treatment leadsto a marked shift from conjugation to N-oxidation. However,N-hydroxy-4-ABP (in contrast to N-hydroxy-2-NA) is mostly releasedfrom hepatocytes in the unconjugated form.     

Comparisons of the effects of chemical carcinogens in mixed cultures of rat hepatocytes and human fibroblasts

Unscheduled DNA synthesis (UDS) was measured simultaneouslyin rat hepatocytes and human fibroblasts when combined culturesof the 2 cell types were exposed to procarcinogens. Human fibroblastswere preincubated with 5-bromo-2'-deoxyuridine (BRdU) to substitutefor thymidine in the DNA. Hepatocyte DNA was separated fromthe heavier BRdU-substituted fibroblast DNA by isopycnic centrifugationsin neutral cesium chloride and the specific activities of theDNA's were determined. In the presence of hepatocytes, benzo[a]pyrene(BP) induced more UDS in the fibroblasts than in the hepatocytes.BP induced no UDS in the fibroblasts in the absence of hepatocytes.Diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) stimulateda significant amount of UDS only in the hepatocytes. Thus, theco-cultures of hepatocytes and fibroblasts responded with UDSto these chemical carcinogens in a manner that parallels thetissue specificity of the carcinogenicity of these chemicalsin vivo. That is, the known hepatocarcinogens DEN and AAF, onlystimulated significant UDS in the hepatocytes, whereas the non-hepatocarcinogen,BP, though activated in these cultures mainly by the hepatocytes,stimulated more UDS in the fibroblasts than in the hepatocytes.The amount of [³H]BP bound to DNA was investigated in the co-culturesand in cultures of fibroblasts alone. When co-cultures wereexposed to [³H]BP the fibroblast DNA had approximately 3 timesmore BP bound to it (per µg DNA) than did the hepatocyteDNA. The amount of [³H]BP bound to the DNA of cultures of fibroblastsalone was 29% of the amount bound to the DNA of the fibroblastsfrom the co-cultures. Thus, although the hepatocytes were mainlyresponsible for the activation of BP, more [³H]BP was boundto the fibroblast DNA. It is suggested that the intercellulardistribution of carcinogenic metabolites may be a significantdeterminant of the carcinogenic effect.     

Inhibition of sulfotransferase in primary cultures of human hepatocytes affecting metabolism and binding of 2-acetylaminofluorene.

The metabolism and DNA binding of acetylaminofluorene (AAF) was investigated in human hepatocytes that were isolated from donor liver tissue by collagenase perfusion. Hepatocytes were treated with 0.01 microM pentachlorophenol (PCP), as a sulfotransferase inhibitor, to investigate the role of sulfotransferase in human bioactivation of aromatic amines. Concentrations of PCP greater than 0.1 microM resulted in cytotoxicity as noted by detachment of cells and atypical morphology. The metabolites of AAF were identified by HPLC as aminofluorene, 7-OH-AAF, 9-OH-AAF, 5-OH-AAF, N-OH-AAF, 1-OH-AAF and 3-OH-AAF. No consistent alteration in the metabolites produced occurred with PCP treatment compared to controls. PCP treatment increased total DNA binding of AAF metabolites compared with controls, suggesting that sulfotransferase does not activate AAF in human hepatocytes. Inhibition of sulfotransferase in human hepatocytes does not decrease DNA binding of AAF metabolites as noted previously with rat hepatocytes. Therefore, PCP may inhibit a detoxication pathway. This study supports N,O-acyltransferase as the critical enzyme for the formation of the major reactive metabolite in human liver.     

Covalent binding to DNA and mutagenicity of 2,4-diaminotoluene metabolites produced by isolated hepatocytes and 9000 g supernatant from Fischer 344 rats

Primary cultures of hepatocytes from adult male F344 rats wereused to investigate the activation of the hepatocarcinogen 2,4-diaminotoluene(2,4-DAT) to metabolites which bound covalently to DNA. Covalentbinding of 2,4-DAT to DNA was significantly greater than thatof a non-carcinogenic isomer, 2,6-DAT. Treatment of male ratswith 5,6-benzo-flavone (BNF) an inducer of cytochrome P-450cand P-450d, had no effect on the binding of 2,4-DAT to DNA ofhepatocytes from these animals. However, treatment of hepatocytesin vitro with metyrapone or piperonyl butoxide, two generalinhibitors of P-450 enzymes, inhibited the binding of 2,4-DATto DNA by {small tilde}80–85%. Two inhibitors of sulfation,pentachlorophenol and 2,5-dichloro-4-nitrophenol, also inhibitedDNA binding in hepatocytes from both BNF-induced (91 and 85%respectively) and control rats (82 and 41% respectively), indicatingthat suffation may also be required. 2,4-DAT was a more potentmutagen than 2,5- or 2,6-DAT in the Ames Salmonella mutagenesisassay using hepatic S9 fractions from F344 rats as an activatingsystem. In contrast to DNA binding, activation of 2,4-DAT tomutagens by S9 fractions from BNF treated rats was greater thanthat by S9 from control rats. The present study shows that 2,4-DATis activated by hepato cytes of F344 rats to products whichbind covalently to DNA. Both cytochrome P-450 and sulfationappear to be involved in the activation.     

Processing of 2-aminofluorene and 2-acetylaminofluorene DNA adducts in Chinese hamster ovary cells

The effects of 2-aminofluorene (AF) DNA damage on cyto-toxicityand DNA-mediated genetic transformation were investigated inChinese hamster ovary (CHO) cells. N-Acetoxy-2-acetylaminofluorene(NA-AAF) treatment of DNA repair-proficient AT3–2 cellsand UVL-10, a UV-hyper-sensitive mutant cell line derived fromAT3–2, showed that UVL-10 cells were markedly more sensitivethan AT3–2 cells to NA-AAF cytotoxicity. Analysis of cellularDNA from NA-AAF-treated cell cultures showed that AF was thepredominant DNA adduct formed in both cell lines, while formationof 2-acetylaminofluorene (AAF) DNA adducts was not detectedin cellular DNA samples of either cell line. Analysis of AFadduct removal showed that kinetics and extent of AF removalwere similar in both cell lines. The effects of cellular processingof AAF DNA adducts in CHO cells were examined by introducingplasmid pSV2gpt DNA containing AAF damage into AT3–2 andUVL-10 cell lines by transfection. For comparative purposes,AF-containing pSV2gpt was also used in parallel experiments.In transfection experiments with AAF-containing pSV2gpt DNA,yields of gpt⁺ transformants declined relative to control frequenciesin a much more pronounced manner in repair-deficient UVL-10cells than in repair-proficient AT3–2 cells. In contrast,transfection with pSV2gpt DNA containing AF adducts had no apparenteffect on transformation frequencies in either cell line, evenat very high levels of modification. Results of co-transformationexperiments in which transfected AAF-containing pSV2gpt DNAmolecules were not subjected to selection for phenotype showedthat in repair-deficient UVL-10 cells, AAF damage in pSV2gptapparently interfered with the ultimate association of transfectedDNA with recipient cell DNA.     

Metabolism and activation of the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine by isolated hepatocytes and pancreatic cells of the Syrian hamster

The metabolism of the pancreatic carcinogen N-nitroso-bis(2-oxopropyl)amine (BOP) was studied using primary hepatocytes and acinar and duct cells isolated from Syrian hamsters. Metabolic activation of BOP was verified by detecting its conversion to CO2 covalently bound metabolites and soluble products containing the alpha-carbon of the nitrosamine. At concentrations below 0.2 mM, BOP was completely activated by hepatocytes within 60 min. At high substrate concentration (1 mM) or high cell density (5 x 10(6) cells/ml), reduction of BOP to N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine and N-nitrosobis(2-hydroxypropyl)amine contributed significantly to the metabolic profile. The conditions which favored metabolic activation of BOP were used to compare metabolism by hepatocytes and pancreatic cells. Under such conditions, the ratio of activation products formed by hepatocytes versus those formed by acinar cells was 14.5:1; the corresponding ratio for covalently bound metabolites was 19:1. Hepatocytes activated BOP 106 times more rapidly than duct cells as determined from yields of activation products or 152 times more rapidly as determined from labeling of cellular macromolecules. Acinar cells showed a higher capacity for metabolic activation than duct cells. The ratio for the yield of activation products from acinar versus duct cells was 4.3:1; the corresponding ratio for covalent binding was 5.8:1. The relatively low capacity of pancreatic cells for activation of BOP compared to hepatocytes is in agreement with the low levels of DNA binding in the pancreas compared to other organs after administration of BOP to the hamster in vivo. The observation that the ratio for total covalent binding of BOP in hepatocytes versus acinar cells was higher than that seen previously for the liver versus the pancreas in vivo is consistent with the hypothesis that alkylating agents derived from BOP reach the pancreas after formation in other organs. The liver would be the prime source for such alkylating agents.     

Modification of 7,8-benzoflavone metabolism in hamster liver and kidney microsomes by hepatic tumor inducing treatments

The effect of pre-treatment of male Syrian golden hamsters with 7,8-benzoflavone (BF), with diethylstilbestrol (DES) and with BF plus DES on the metabolism of [14C]BF in hepatic and renal microsomes has been studied in vitro. Whereas hepatic microsomes from DES-treated animals produced the same pattern of BF metabolites as control microsomes, a marked quantitative and qualitative alteration of BF metabolism was observed with liver microsomes from animals pre-treated with BF and with BF plus DES: the metabolic rate was increased and three new metabolites were formed which were not observed with control hepatic microsomes. These metabolites, which were tentatively identified as BF-5,6-dihydrodiol and two isomeric dihydroxy-BFs, were not detected in incubations with renal microsomes under any pre-treatment regimen. Non-extractable binding of radioactivity to hepatic and renal microsomal protein was observed in all incubations but did not exhibit as pronounced a dependence on pre-treatment as did the pattern of BF metabolites. Based on the metabolic data it is concluded that BF induces its own oxidative metabolism. Among the metabolites are reactive intermediates that bind to cellular macromolecules and may play an important role in tumor formation in the male Syrian hamster liver following prolonged treatment with BF plus DES.     

Human and rat kidney cell metabolism of 2-acetylaminofluorene and benzo(a)pyrene

The metabolism and mutagenic activation of the model carcinogens benzo(a)pyrene [B(a)P] and 2-acetylaminofluorene (AAF) by human and rat kidney cells were measured. A slicing technique followed by enzyme digestion was utilized to obtain the kidney cells. Although levels of total metabolism of B(a)P by rat and human kidney cells were similar, analysis of specific metabolites of B(a)P indicated that species differences existed. Human kidney cells produced the organic-soluble metabolites B(a)P-9,10-diol, B(a)P-4,5-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, and B(a)P-9-phenol. Rat kidney cells produced organic-soluble B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-4,5-diol, and B(a)P-6,12-quinone. Both species produced sulfate and glucuronide conjugates of all products. For AAF, kidney cells from some human tissues produced up to four times the level of total metabolites compared to rat kidney cells. Organic-soluble metabolites were qualitatively similar between the species and consisted of 2-aminofluorene (AF), N-hydroxy-AAF and ring-hydroxylated products at the 1, 3, 5/9, 7, and 8 positions. Sulfate and glucuronide conjugates of these metabolites were also detected. Human interindividual variation with kidney cells was about 2.5-fold for total AAF metabolism and up to 6-fold for individual AAF metabolites. For B(a)P metabolism, human interindividual variation in total metabolism was low while for specific metabolites there was up to a 4-fold variation. Levels of AAF and AF cell-mediated Salmonella typhimurium mutagenesis were significantly higher with human cells as compared to rat kidney cells. It appears that the differences between human and rodent kidney cell metabolism of chemical carcinogens vary with the chemical class and understanding these differences will be necessary in the extrapolation of rodent carcinogenesis data to humans.     

Correlation between induction of unscheduled DNA synthesis in the liver and excretion of mutagenic metabolites in the urine of rats exposed to the carcinogenic air pollutant 2-nitrofluorene

The genotoxic effects of 2-nitrofluorene (NF) have been studiedin vivo by measuring the induction of DNA repair, i.e. unscheduledDNA synthesis (UDS), in hepatocytes from male rats pretreatedby oral gavage with NF. During the NF exposure, Urine was collectedfor 24 h, and its mutagenicity was investigated in the plateincorporation assay, using Salmonella TA98 as tester strain.The urine samples were also used for the identification of excretedmetabolites of NF. Rats treated with 2-acetylaminofluorene (AAF)were studied simultaneously. A positive UDS response was observed12 and 24 h following a single gavage exposure to 12.5, 25 and50 mg/kg NF, with the response returning to near control levelsby 36 h. The positive control AAF induced approximately twicethe response observed with NF, and both compounds gave a UDSresponse that was 2–3 times higher in Wistar rats relativeto Sprague-Dawley rats. A potent directacting mutagenic effectwas observed in urine samples after NF treatment, while AAFexposure only gave rise to a weak mutagenic effect, the NF/AAFratio being 10/1. The stronger urinary mutagenicity after NFtreatment relative to AAF treatment was associated with thepresence of hydroxylated NFs. The genotoxic effect observedin the liver after NF treatement is, on the other hand, morelikely due to the same AAF metabolites that are also formedafterin vivo treatment with AAF.     

Comparison of human and rat hepatocyte metabolism and mutagenic activation of 2-acetylaminofluorene

A method has been developed to assess the metabolism and mutagenic activation of carcinogens using human and rodent hepatocytes in vitro. A slicing technique which was especially useful for nonperfusable biopsy and resected surgical human liver tissue was used to prepare the hepatocytes. Metabolites of the model carcinogen 2-acetylaminofluorene (AAF) produced by human and rat hepatocytes were similar and consisted primarily of 2-aminofluorene with ring hydroxylated products at the 1-, 3-, 5/9-, 7-, and 8-positions produced in addition to N-hydroxy-AAF. Sulphate and glucuronide conjugates of ring-hydroxylated metabolites and 2-aminofluorene were detected. Metabolism and cell-mediated Salmonella mutagenicity illustrated interindividual variation with human hepatocytes. Levels of metabolism and mutagenesis were generally higher with human hepatocytes compared to rat hepatocyte results. The increased levels of metabolism and mutagenesis of AAF by human hepatocytes compared to rat hepatocytes probably indicates a different sensitivity to hepatocarcinogenic effects of AAF on humans as compared to rats. Understanding differences and similarities between human and rodent carcinogen activation capabilities should be useful in the extrapolation of rodent carcinogenesis data to humans.     

Comparison of the metabolic profiles of benzo[alpha]pyrene obtained from primary cell cultures and subcellular fractions derived from normal and methylcholanthrene-induced rat liver

Hepatocyte primary cell (HPC) cultures derived from either (a) non-induced (normal) or (b) methylcholanthrene (MC)-induced rat liver actively metabolized the carcinogen benzol[alpha]pyrene (BaP) over a 24-h period. In both cases, the BaP metabolites generated were qualitatively similar to those seen in the metabolism of BaP by isolated rat liver microsomal fractions; in addition, unidentified compounds were evident in the chromatographic profile generated by the cultured cells. In cells derived from (a), levels of known metabolites (phenols and diols) increased over the time period studied. On the other hand, in cells derived from (b), levels of diols decreased markedly after 8 h. These results suggest that induction with MC enhances both activation and, to a greater extent, conjugative-detoxification pathways of BaP, so that in cells obtained from (b) the formation of water-soluble metabolites is enhanced and levels of organic soluble metabolites are lower than in cells obtained from (a). Metabolism of BaP in primary cell culture derived from rat liver is thus seen to be similar to in vivo metabolism of the carcinogen, but somewhat in contrast to the in vitro microsomal (subcellular) metabolism of BaP where conjugative-detoxification pathways are virtually inoperative.     

Induction of the aromatic hydrocarbon receptor by trans-4-acetylaminostilbene in rat liver. Comparison with other aromatic amines

The effect of the aromatic amines 2-acetylaminofluorene (AAF),2-acetylaminopbenanthrene (AAP) and trans-4-acetyl-aminostilbene(AAS) on the rat hepatic aromatic hydrocarbon (Ah) receptorlevel was studied. 3-Methylcholanthrene (MC), as a known receptorligand, was used as a control. The complete liver carcinogenAAF and MC did not alter significantly the hepatic receptorconcentration. In contrast, the strong liver tumor initiatorAAS doubled the hepatic Ah receptor level when a dose of 20µmol/kg was administered for 5 days. AAP increased theamount of the receptor 1.5-fold.     

Uptake of N-nitrosobis(2-oxopropyl)amine by isolated rat and hamster hepatocytes: species differences and evidence for an active carrier-mediated transport process

The rates of uptake of the carcinogen N-nitrosobis(2-oxo-propyl)amine(BOP) by hepatocytes isolated from Fischer rats and Syrian hamsterswere determined in order to investigate species differencesin cellular transport of the carcinogen. Initial rates of uptakeof (1-¹⁴C)BOP by hepatocytes were measured using a rapid centrifugationtechnique. At cell densities from 1.5 to 6 x 10⁶ cells/ml, initialrates of uptake were as much as 4-fold more rapid in hamsterhepatocytes than in those of the rat. The cell/medium distributionratio for hamster hepatocytes reached a value of 9.0 after a20-min incubation with an extracellular BOP concentration of20 µM. Under the same conditions, the cell/medium distributionratio for rat hepatocytes was only 2.4. These results indicatedthat BOP uptake proceeded against a concentration gradient andwas more rapid in hamster hepatocytes. In both species, therates of uptake were saturable with increasing concentration(2–685 µM) and displayed biphasic kinetics characteristicof high-affinity (Km < 20 µM) and low-affinity (Km30 µM) process for the uptake of BOP. Evidence for theinvolvement of an ATP-dependent active carrier-mediated transportprocess was obtained from experiments in which hepatocytes werepreincubated with metabolic inhibitors. Significant inhibitionof uptake was observed in the presence of KCN, carbonyl cyanide-3-chlorophenylhydrazone,antimucin A, oligomycin and other agents which interfere withelectron transport or ATP generation. Based on the reductionin uptake rates, rat hepatocytes were more sensitive to theeffects of these inhibitors. These results suggest that theentry of BOP into hepatocytes is under cellular regulation andthat the more rapid rate of uptake in liver cells of the hamstermay be one factor responsible for the observation that BOP isa more potent hepatotoxin and carcinogen in this species.