Quantitative assessment of enzyme induction by peroxisome proliferators and application to determination of effects on triglyceride biosynthesis in primary cultures of rat hepatocytes

Potencies for the induction of peroxisomal fatty acyl-CoA oxidase (FACO) and microsomal laurate hydroxylase (LH) were determined for clofibric acid (CPIB), ciprofibrate (Cipro) and gemfibrozil (Gem) in primary cultures of rat hepatocytes based on complete concentration-response analysis and determination of theoretical maximum inductive responses for Cipro. CPIB and Cipro each induced FACO and LH in a concentration-dependent manner. Scatchard analysis of the data allowed calculation of EC50 values (mM) of 0.82 and 0.028 (for FACO) and 0.22 and 0.0081 (for LH) for CPIB and Cipro respectively. The EC50 ratios (CPIB/Cipro) were identical (29-fold) for induction of FACO and LH, supporting the concept that these enzymes are induced by CPIB and Cipro through a common mechanism. By comparison, Gem was relatively ineffective as an inducer of FACO and LH. Furthermore, Gem did not antagonize Cipro-mediated enzyme inductions, suggesting that Gem is a peroxisome proliferator of low potency rather than a partial agonist. Based on the potency and time-course profiles observed for induction of FACO and LH, the effects of CPIB, Cipro and Gem on triglyceride (TG) biosynthesis were determined in the cultured rat hepatocytes. Conditions of maximal FACO and LH induction by the drugs did not result in inhibition of TG biosynthesis in the cells. These results support the in vivo evidence which indicates that FACO and LH induction are not causally linked to the hypotriglyceridemic actions of peroxisome proliferating drugs.     

Comparison of the effects of nafenopin on hepatic peroxisome proliferation and replicative DNA synthesis in the rat and Syrian hamster.

Male Sprague-Dawley rats were fed control or 0.1% nafenopin diet and male Syrian hamsters were fed control or 0.25% nafenopin diet for periods of 7 and 54 days. Nafenopin treatment produced a sustained increase in liver weight and induction of hepatic peroxisomal and microsomal fatty acid-oxidizing enzyme activities, with a greater effect being observed in the rat. Replicative DNA synthesis was studied by implanting osmotic pumps containing [3H]thymidine during study days 0-7 and 47-54. Cell replication, determined either as the hepatocyte labelling index or by incorporation of radioactivity into liver whole homogenate DNA, was increased in rats given nafenopin for 7 and 54 days. In contrast to the rat, no significant effect on replicative DNA synthesis was observed in the Syrian hamster. These results provide further evidence for species differences in hepatic peroxisome proliferation, with the Syrian hamster being less responsive than the rat. Furthermore, while peroxisome proliferators produce hyperplasia in rat and mouse liver, these data suggest that they may not have any marked effect on hepatic replicative DNA synthesis in the Syrian hamster.     

Inhibitory effects of dexamethasone on hepatocyte growth factor-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

We investigated the effects of dexamethasone on hepatocyte growth factor (HGF)-induced DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. Isolated hepatocytes were cultured at a density of 3.3 × 10(4) cells/cm(2) in Williams' medium E containing 5% newborn bovine serum and various concentrations of dexamethasone for 1, 2, and 3 h. After a 3-h attachment period, the medium was then changed, and cells were cultured in serum-free dexamethasone (10(-10) M)-containing Williams' medium E with or without glucocorticoid receptor antagonists. After addition of dexamethasone to the culture medium, the growth-stimulating effects of HGF (5 ng/mL) on the primary cultured hepatocytes were time- and dose-dependently inhibited. The mineralcorticoid aldosterone (10(-7) M) did not produce the same growth-inhibitory effects as dexamethasone (10(-8) M). The inhibitory effects of dexamethasone were reversed by treatment with the glucocorticoid-receptor antagonist mifepristone (RU486, 10(-6) M) or a monoclonal antibody against glucocorticoid receptor (100 ng/mL). In addition, the growth-inhibitory dose of dexamethasone did not affect HGF-induced receptor tyrosine kinase and extracellular signal-regulated kinase 2 phosphorylation. These results indicate that dexamethasone dose-dependently delays and inhibits HGF-induced DNA synthesis and proliferation through its own intracellular receptor in primary cultures of adult rat hepatocytes.     

Background DNA repair synthesis in rat hepatocyte cultures used for genotoxicity testing

DNA repair synthesis in primary rat hepatocyte cultures (HPC) was investigated using the bromodeoxyuridine (BrdUrd) density-shift method and autoradiography. Analysis by density-gradient centrifugation of DNA labelled with BrdUrd and [³H]deoxycytidine ([³H]dCyd) for 18–20 hr showed that considerable DNA repair replication occurs in HPC even in the absence of an added genotoxic agent. Repair was demonstrated to be most probably a consequence of DNA damage caused by the collagenase perfusion of the liver during hepatocyte isolation, and by β-decay of the ³H-labelled DNA precursor. Autoradiographic analysis of the distribution of silver grains over nuclei in intact non-S-phase cells, and over isolated nuclei from HPC exposed to [³H]dCyd for 18–20 hr, showed that the vast majority of the radioactivity was incorporated into the nuclei themselves, and not into overlying cytoplasm or mitochondria. In addition, direct localization of mitochondria in hepatocytes using the mitochondrion-specific dye rhodamine 123 showed that very few mitochondria were actually located over the nucleus. The results suggest that cytosolic labelling of control HPC in autoradiographs is mainly caused by mitochondrial DNA synthesis whereas nuclear labelling essentially reflects repair synthesis. They call into question the commonly applied practice of evaluating unscheduled DNA synthesis (UDS) in HPC by subtracting the number of cytosolic silver grains from nuclear grains and expressing repair synthesis as net grains per nucleus.     

Comparison of cytochrome P450 isoenzyme profiles in rat liver and hepatocyte cultures. The effects of model inducers on apoproteins and biotransformation activities

The metabolic profile of seven subfamilies of cytochrome P450 (P450IA, IIA, IIB, IIC, IIE, IIIA, IVA) was studied in rat liver (in vivo) and in primary hepatocyte cultures (in vitro) after treatment with various inducers. The dealkylation of 7-ethoxyresorufin (EROD) and 7-pentoxyresorufin (PROD), aniline 4-hydroxylation and the regio- and stereoselective hydroxylation of testosterone were measured to characterize the isoenzyme pattern in intact hepatocytes and in liver microsomes. Occurrence of isoenzyme apoproteins was determined using Western blotting. Primary cultures of rat hepatocytes retain the capacity to respond to inducers of isoenzymes belonging to six different subfamilies (P450IA, IIA, IIB, IIC, IIIA and IVA). Treatment of cells with beta-naphthoflavone revealed a P450-activity profile similar to in vivo, namely a highly induced EROD (P450IA1), a small enhancement of testosterone 7 alpha-hydroxylation (P450IIA) and a marked reduction in 2 alpha- and 16 alpha-hydroxylation (P450IIC11). Exposure of cultured cells to phenobarbital resulted in a higher testosterone 16 beta-hydroxylation (reflecting P450IIB), though to a lesser extent than in vivo. The induction of P450IIIA due to both phenobarbital and dexamethasone, as mirrored by 6 beta- and 15 beta-hydroxylation of testosterone, was the same in cultured hepatocytes and in vivo. Treatment of cells with clofibric acid resulted in an induction profile similar to the one observed in liver microsomes from clofibrate-treated rats: the apoprotein P450IVA as well as the apoprotein P450IIB1/2 and its associated activities (PROD and testosterone 16 beta-hydroxylation) were induced. Isoniazid, a known in vivo inducer of P450IIE1 and aniline 4-hydroxylation, did not change any of the determined P450-dependent activities in vitro.     

Peroxisome proliferators induce apoptosis and decrease DNA synthesis in hepatoma cell lines

We examined the effects of various peroxisome proliferators (PPs) such as the hypolipidaemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO) and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on peroxisomal enzyme activities, apoptosis and DNA synthesis in rat FaO and human HepG2 hepatoma cell lines. Both growing and confluent cultures were treated with PPs (250 microM) for 48 or 72 h. In accordance with our previous observations in PP-treated primary hepatocyte cultures of rat and human origin, the various PPs increased peroxisomal enzyme activities in rat FaO cells but not in human HepG2 cells. PPs strongly induced apoptosis in FaO cells. They did not affect TGFbeta-induced apoptosis, with the exception of DEHP and NAFE, respectively blocking and increasing induced apoptosis in confluent cultures. Moreover, PPs produced a minor, but significant, decrease in DNA synthesis in FaO cells. PPs also decreased DNA synthesis in growing HepG2 cells, and CLO, CIPRO and NAFE induced apoptosis in confluent HepG2 cultures. This is in opposition with the effects of PPs on primary hepatocyte cultures, i.e. inhibition of both spontaneous and TGFbeta-induced apoptosis and increases in DNA synthesis in rat hepatocytes, and unchanged mitosis-apoptosis balance in human hepatocytes.     

The effect of beclobric acid and clofibric acid on peroxisomal beta-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes

The peroxisome-proliferating effects of clofibric acid and beclobric acid were studied in primary cultures of hepatocytes derived from rat, monkey (Macaca fascicularis) and human liver. Determination of peroxisomal fatty acid beta-oxidation and morphometrical analysis of the peroxisomal compartment were performed after incubation of 1-day-old hepatocyte cultures for 3 days with either compound. In rat liver cell cultures both compounds gave a 10-fold increase in peroxisomal beta-oxidation, a 3-fold increase in the relative number of peroxisomes and a 1.5-fold increase in the mean size of peroxisomes. Beclobric acid gave its maximal effect at a concentration of 10 microM, which is at least one order of magnitude lower than the maximum-effect concentration of clofibric acid. At concentrations greater than 300 microM beclobric acid was cytotoxic. No stimulation of peroxisomal fatty acid beta-oxidation was found in either monkey or human hepatocyte cultures. Morphometrical analysis also showed no increase in the peroxisomal compartment in cultures derived from these species, as indicated by the lack of increase in both relative number and size of peroxisomes. In all three species tested beclobric acid was equally cytotoxic for hepatocytes in vitro. These results are of relevance for the interpretation of the peroxisome-proliferating effects of clofibrate and similar compounds in rats. Since peroxisome proliferation may be correlated to increased hepatic tumour incidences in the rat, the absence of peroxisome proliferation in primates suggests the absence of tumourogenic activity by hypolipidemic compounds in these species.     

Effect of the histone deacetylase inhibitor trichostatin A on spontaneous apoptosis in various types of adult rat hepatocyte cultures

Acetylation and deacetylation of histones, catalysed by histone acetyl transferases and histone deacetylases (HDAC), respectively, are known to be involved in gene expression regulation. Here, the effect on the activity and expression of several apoptosis-related proteins of trichostatin A (TSA), a well-known HDAC inhibitor, were studied in short-term (conventional monolayer) and long-term cultured (collagen I gel sandwich cultures and co-cultures) adult rat hepatocytes. No significant effects of TSA on the caspase-3-like activity were seen in rat hepatocytes cultured in a sandwich configuration or in a co-culture with rat liver epithelial cells of primitive biliary origin. In both culture models, the basal level of apoptosis was found to be much lower than in control monolayer cultures. In the latter system, it was found that, after 4 days of culture, TSA decreased the levels of caspase-3 (both proform and p17 fragment) and of the pro-apoptotic protein Bid. No effect of TSA was found on the expression of Bax. As expected, a TSA-mediated increase of acetylated histones H3 and H4 was observed in all culture systems examined. In addition, in the presence of TSA, increased albumin secretion and cytochrome P450 1A1/2 and 2B1-dependent enzyme activities were found in conventional cultures after 7 days. In conclusion, TSA delayed the occurrence of apoptosis and loss of liver specific functions in conventional hepatocyte monolayers. In contrast, in hepatocyte culture models in which spontaneous apoptosis is already minimised through the addition of either extracellular matrix components (sandwich cultures) or non-parenchymal liver cells (co-cultures), TSA did not have any additional anti-apoptotic effect.     

Mechanisms of cyclosporine A-induced apoptosis in rat hepatocyte primary cultures

In rat hepatocytes and isolated liver mitochondrial fractions, Cyclosporine A (CsA) is often used as a specific inhibitor of mitochondrial Ca(2+) release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 h. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 h with CsA at concentrations of 0, 10, 25, and 50 microM. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3, and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation and the number of TUNEL- and Annexin V-positive cells increased dose-dependently without any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 h of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEID-CHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events that trigger the apoptosis cascade. Transmission electron microscopy confirmed an increase in the number of necrotic cells after 20 h, but not after 4 h, compared with controls.     

Induction of peroxisomal acyl CoA oxidase activity and lipid peroxidation in primary rat hepatocyte cultures

Peroxisome proliferators have been suggested to induce liver carcinogenesis as a result of increased peroxisomal hydrogen peroxide production and cellular oxidative stress. Primary monolayer cultures of hepatocytes isolated from male F344 rats were incubated in medium containing one of three different peroxisome proliferators and examined for the induction of peroxisomal CoA oxidase activity and lipid peroxidation. The latter parameter was determined by measuring levels of conjugated dienes in lipid fractions extracted from harvested cells. The peroxisome proliferators used in these studies were nafenopin and clofibric acid (two hypolipidemic drugs) and mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the industrial plasticizer, di(2-ethylhexyl)phthalate (DEHP). The relative specific activity of peroxisomal acyl CoA oxidase was increased by about 300% after incubation for 44 h with 200 microM nafenopin; lower levels of induction were observed with clofibric acid or MEHP. Relative to controls, the level of conjugated dienes was increased approximately 2-fold after incubation with 200 microM nafenopin; there was no apparent increase in conjugated dienes after incubation with up to 200 microM MEHP or 400 microM clofibric acid. The increase in conjugated dienes with 200 microM nafenopin was inhibited by co-incubation with the antioxidant, N,N'-diphenyl-p-phenylenediamine. Thus, peroxisomal enzyme induction by nafenopin can result in membrane lipid peroxidation and monolayer cultures of rat hepatocytes may provide a useful model system for studying relationships between peroxisome proliferation, enhanced hydrogen peroxide production and cellular changes due to hepatic oxidative stress.     

Effects of hepatic peroxisome proliferators and 12-O-tetradecanoyl phorbol-13-acetate on catalase and other enzyme activities of embryonic cells in vitro

The effects of the hepatic peroxisome proliferators (HPPs) clofibrate, di-(2-ethylhexyl)-phthalate (DEHP), mono-(2-ethylhexyl)phthalate (MEHP) and 2,4-dichlorophenoxy acetic acid (2,4-D) on the activities of some peroxisome-associated enzymes and marker enzymes for other organelles, have been studied in primary Syrian hamster embryo (SHE) cells and Wistar rat embryo (WRE) cells. The majority of the cells are fibroblast-like. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) was included as it has been suggested that it may act as a peroxisome proliferator. The specific activities of catalase, fatty acyl-CoA oxidase (FAO) and peroxisomal beta-oxidation were approximately 100-fold lower in the embryonic cells than in rat hepatocytes. Other peroxisome-associated oxidases were not detected. The dihydroxyacetone-phosphate acyltransferase (DHAPAT) activity was comparable to that in rat liver. Marker enzymes for other organelles had specific activities comparable to rat hepatocytes. Catalase was shown by digitonin titration to be contained in a peroxisome-like compartment in both SHE and WRE cells. Clofibrate, DEHP and MEHP increased the catalase activity, which might suggest peroxisome proliferation. However, the findings that FAO and peroxisomal beta-oxidation did not increase or only very slightly, argue against peroxisome proliferation. 2,4-D and TPA induced no or only a very slight increase in the catalase activity.     

The absence of testicular atrophy and in vivo and in vitro effects on hepatocyte morphology and peroxisomal enzyme activities in male rats following the administration of several alkanols

Previous studies have shown that ethylhexanol (2-EH) and its oxidation products, but not n-hexanol, produce hepatomegaly, peroxisomal proliferation and hypotriglyceridaemia. In the present studies we have confirmed that at 1 mmol/kg doses, neither the linear nor branched chain alcohols induce testicular atrophy, hepatomegaly, peroxisome proliferation or hypolipidaemia. In vivo, neither the free alcohols nor their metabolic products seem to be responsible for the activity of the parent plasticiser. The released monoesters are probably the more potent metabolic products responsible for the hepatomegaly, peroxisomal proliferation and hypolipidaemia. This contention is supported by the in vitro hepatocyte data which demonstrate the induction of peroxisomal oxidative enzymes by MEHP whereas the alcohols were without effects.     

Xenobiotic metabolizing enzyme activities in rat, mouse, monkey, and human testes.

The capacity of the testis to metabolize xenobiotics has been proposed to play a role in the susceptibility of different species to testicular toxicity. Since species differences in testicular xenobiotic metabolizing enzyme activities are not well documented, the primary objective of the present study was to compare enzyme activities in subcellular fractions prepared from rat, mouse, monkey, and human testes. In microsomal fractions, enzyme activities measured were pentoxyresorufin O-dealkylase (PROD), ethoxyresorufin O-dealkylase (EROD), and epoxide hydrolase (mEH). In cytosolic preparations, epoxide hydrolase (cEH) and glutathione S-transferase (cGST) activities were measured. PROD activity was not detectable in any of the species studied, while it was readily detected in liver microsomes used as a positive control. Although EROD activity was low, it was measurable in testicular microsomes from rat and mouse, but not monkey or human. No marked species differences in cEH activity were found. In contrast, mEH activity was low in the monkey, intermediate in the rat, and highest in the human and mouse. cGST activity was significantly lower in the two primate species compared with the rat and the mouse. The levels of activity of the xenobiotic metabolizing enzymes studied were generally more than an order of magnitude lower in the testis as compared to the liver. However, in rat and mouse, the levels of mEH and cGST activities in testis were relatively similar to hepatic levels. Overall, these data indicate that species differences in capacity to metabolize xenobiotics may play a role in differential sensitivity to testicular toxicants.     

Lack of effect of coumarin on unscheduled DNA synthesis in the in vivo rat hepatocyte DNA repair assay.

The ability of coumarin to induce UDS in male Sprague-Dawley CD rat hepatocytes in vivo was assessed using the unscheduled DNA synthesis (UDS) assay. From a preliminary toxicity study the oral maximum tolerated dose (MTD) of coumarin was determined to be 320 mg/kg body weight. For the UDS studies, rats were treated with 0 (corn oil control), 32 (one-tenth the MTD), 107 (one-third the MTD) and 320 (MTD) mg/kg coumarin via oral gavage. Rats were also treated with 20mg/kg body weight dimethylnitrosamine (DMN) or 50mg/kg body weight 2-acetylaminofluorene (2-AAF) as positive controls for the 2-4 hr and 12-16 hr expression of UDS, respectively. Hepatocytes were isolated by liver perfusion either 2-4 hr or 12-16 hr after treatment and cultured in medium containing [methyl-(3)H]thymidine for 4 hr and assessed for UDS by grain counting of autoradiographs. Coumarin treatment at doses of 32-320 mg/kg body weight had no statistically significant or dose-related effect on UDS in rat hepatocytes either 2-4 hr or 12-16 hr after dosing. In contrast, both DMN 2-4 hr after dosing and 2-AAF 12-16 hr after dosing produced significant increases in UDS assessed as the net nuclear grain count. Both genotoxins also increased the percentage of hepatocyte nuclei with greater than 5 net grains. Treatment with coumarin, DMN and 2-AAF had no statistically significant effect on the proportion of rat hepatocytes undergoing replicative DNA synthesis. In summary, this study demonstrates that coumarin does not induce UDS in hepatocytes of male Sprague-Dawley CD rats after oral administration at doses up to the MTD of 320 mg/kg. The responsiveness of the animals used in this study to genotoxic agents was demonstrated by the clear induction of DNA repair after treatment with DMN and 2-AAF.     

高糖对人肾小球系膜细胞过氧化物酶体增殖物激活受体家族表达的影响

目的研究高糖作用下人肾小球系膜细胞(HMC)中过氧化物酶体增殖物激活受体(PPAR)家族的表达及转录活性的变化,探讨PPAR介导的信号转导途径在糖尿病性肾病发生发展中的作用。方法使用3层滤网法分离HMC,3～7代细胞用于实验。HMC分为正常对照组(5mmol.L-1葡萄糖,NG)、渗透浓度对照组(5mmo.lL-1葡萄糖+25mmol.L-1甘露醇,MG)和高糖组(30mmol.L-1葡萄糖,HG)。mRNA水平及蛋白水平的检测分别采用实时定量PCR和蛋白印迹法。结果NG组HMC中PPARα,PPARγ及PPARδ mRNA均有表达,PPARγ蛋白亦有表达。MG组上述mRNA表达无明显变化。高糖刺激后,PPARα mRNA表达明显高于MG组,PPARγ和PPARδmRNA表达减少,PPARγ蛋白表达亦明显降低,PPARγ激活的基因adipophilin的表达亦减少。结论高糖能上调PPARα基因的表达,抑制PPARγ和PPARδ基因表达,并降低PPARγ的转录活性,提示高糖可能通过PPAR介导的信号转导途径在糖尿病性肾病的发生发展中发挥重要作用。     

Thiourea induces DNA repair synthesis in primary rat hepatocyte cultures and gene mutations in V79 Chinese hamster cells

Thiourea was investigated for its capacity to cause DNA alterations in cultured mammalian cells. The induction of DNA repair in primary rat hepatocyte cultures and of gene mutations in V79 Chinese hamster cells were used as biological endpoints. In hepatocytes, thiourea elicited a linear increase in DNA repair replication in the concentration range tested (5–25 mM). In V79 cells, thiourea (10–40 mM) significantly increased the frequency of 8-azaguanine-resistant mutants. The present results show that thiourea is weakly, but definitely, genotoxic and mutagenic in cultured mammalian cells.     

Lack of effect of furfural on unscheduled DNA synthesis in the in vivo rat and mouse hepatocyte DNA repair assays and in precision-cut human liver slices.

The ability of furfural to induce unscheduled DNA synthesis (UDS) in hepatocytes of male and female B6C3F(1) mice and male F344 rats after in vivo administration and in vitro in precision-cut human liver slices has been studied. Preliminary toxicity studies established the maximum tolerated dose (MTD) of furfural to be 320 and 50 mg/kg in the mouse and rat, respectively. Furfural was dosed by gavage at levels of 0 (control), 50, 175 and 320 mg/kg to male and female mice and 0, 5, 16.7 and 50 mg/kg to male rats. Hepatocytes were isolated by liver perfusion either 2-4 h or 12-16 h after treatment, cultured in medium containing [3H]thymidine for 4 h and assessed for UDS by grain counting of autoradiographs. Furfural treatment did not produce any statistically significant increase or any dose-related effects on UDS in mouse and rat hepatocytes either 2-4 h or 12-16 h after dosing. In contrast, UDS was markedly induced in mice and rats 2-4 h after treatment with 20 mg/kg dimethylnitrosamine and 12-16 h after treatment of mice and rats with 200 mg/kg o-aminoazotoluene and 50 mg/kg 2-acetylaminofluorene (2-AAF), respectively. Precision-cut human liver slices from four donors were cultured for 24 h in medium containing [3H]thymidine and 0-10 mM furfural. Small increases in the net grain count (i.e. nuclear grain count less mean cytoplasmic grain count) observed with 2-10 mM furfural were not due to any increase in the nuclear grain count. Rather, it was the result of concentration-dependent decreases in the mean cytoplasmic grain counts and to a lesser extent in nuclear grain counts, due to furfural-induced cytotoxicity. In contrast, marked increases in UDS (both net grain and nuclear grain counts) were observed in human liver slices treated with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 mM aflatoxin B(1) and 0.005 and 0.05 mM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. This study demonstrates that furfural does not induce UDS in the hepatocytes of male and female B6C3F(1) mice and male F344 rats after oral treatment at doses up to the MTDs. Moreover, human liver slice studies suggest that furfural is also not a genotoxic agent in human liver.