DNA damage induced by 3,3'-dimethoxybenzidine in liver and urinary bladder cells of rats and humans.

3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic DMB concentrations ranging from 56 to 180 microM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 microM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 microM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.     

Comparison of the sensitivity of human and rat hepatocytes to the genotoxic effects of metronidazole

Metronidazole (MNZ), an antiprotozoan and antibacterial agent, has been shown to yield DNA-damaging reactive species after nitroreductive biotransformation. The genotoxic effect of MNZ was studied in primary cultures of both rat and human hepatocytes. In millimolar concentrations MNZ produced DNA fragmentation, as measured by the alkaline elution technique, and unscheduled DNA synthesis, as evaluated by quantitative autoradiography, in rat hepatocytes. The amount of DNA damage was directly related to the dose and the length of exposure, was increased by hypoxia and GSH depletion, and was markedly reduced by inhibition of cytochrome P-450 activity. In the same experimental conditions human hepatocytes resulted constantly more resistant than rat hepatocytes to the genotoxic activity of MNZ. These findings suggest that the rat hepatocyte model might be an inappropriate predictor of nitroimidazoles genotoxicity.     

Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes

The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.5 fold and 1.3 fold, respectively, by exposure to Rif at 2 to 50 microM as compared with 0.1% DMSO-treated controls. MRP2 mRNA levels in the same human hepatocytes were significantly increased by 1.2 to 1.8 fold by exposure to Rif at 50 microM as compared with controls. In primary cultures of rat hepatocytes, Mdr1a and Mdr1b mRNA levels were not increased or only slightly increased at 24 hr by exposure to any of the inducers at 2, 10 or 50 microM. Mrp2 mRNA levels in the same rat hepatocytes were significantly increased by 7 to 45 fold by exposure to Dex at 2 microM as compared with controls. Based on the species differences observed in the present study, primary cultures of cryopreserved hepatocytes from both the human and rat should be useful in preclinical drug development for evaluating candidate drugs for transporter induction.     

DNA damage and micronuclei induced in rat and human kidney cells by six chemicals carcinogenic to the rat kidney

Six chemicals, known to induce kidney tumors in rats, were examined for their ability to induce DNA fragmentation and formation of micronuclei in primary cultures of rat and human kidney cells, and in the kidney of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the Comet assay, and in micronuclei frequency, were obtained in primary kidney cells from both male rats and humans of both genders with the following subtoxic concentrations of five of the six test compounds: bromodichlorometane (BDCM) from 0.5 to 4 mM, captafol (CF) from 0.5 to 2 microM, nitrobenzene (NB) from 0.062 to 0.5 mM, ochratoxin A (OTA) from 0.015 to 1.215 microM, and trichloroethylene (TCE) from 1 to 4 mM. Benzofuran (BF), consistent with its carcinogenic activity for the kidney of female, but not of male rats, at concentrations from 0.125 to 0.5 mM gave positive responses in cells from females but did not induce DNA damage and increased the frequency of micronuclei in cells from males to a lower extent; in contrast, it was active in cells from humans of both genders. DNA-damaging and micronuclei-inducing potencies were similar in the two species. In agreement with these findings, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were obtained in the kidney of rats, given p.o. a single dose (1/2 LD50) of the six compounds, BF in this assay being more genotoxic in female than in male rats. Taken as a whole, these findings give further evidence that kidney carcinogens may be identified by short-term genotoxicity assays, using as target kidney cells, and show that the six chemicals tested produce, in primary cultures of kidney cells from human donors, effects similar to those observed in rats.     

DNA adducts in cultures of polychlorinated biphenyl-treated human hepatocytes

Polychlorinated biphenyls (PCBs) are persistent environmental chemicals that accumulate at the apex of food chains. Several scientific committees support its designation as a human carcinogen, even though the precise mechanism of carcinogenesis remains controversial. In view of its uncertain ability to cause DNA damage in human liver, we investigated the effects of Aroclor 1254, Aroclor 1016, and 4-chlorobiphenyl (4-CB) on DNA adduct formation in cultures of primary human hepatocytes from five donors. Based on (32)P-postlabeling assays, we detected DNA adducts in native human liver as well as untreated, i.e., control cultures of human hepatocytes. Treatment of human hepatocytes with Aroclor 1016 and Aroclor 1254 resulted in four-fold (NP1) and seven-fold (butanol) increases in DNA adduct formation. Further, two and six new adduct spots were detected by multidirectional thin-layer chromatography after nuclease P1 and butanol enrichment. Treatment of human hepatocyte cultures with 4-CB led to 209 adducts per 10(9) nucleotides at the 60 microM concentration, and we show metabolically activated PCBs to be more efficient in the production of DNA-binding species compared with higher chlorinated PCB mixtures. Our study is therefore highly suggestive for a link between PCB exposure and DNA insult in human hepatocytes.     

DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by four rat thyroid carcinogens

Four chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measures by the Comet assay, were obtained after a 20-h exposure to the following subtoxic concentrations of the four test compounds: 2,4-diaminoanisole (DAA) from 0.10 to 1.0 mM, 4,4'-methylene-bis(N,N-dimethyl)benzenamine (MDB) from 0.32 to 1.8 mM, propylthiouracil (PTU) from 1.8 to 5.6 mM, and 4,4'-thiodianiline (THA) from 0.032 to 0.18 mM. Under the same experimental conditions, DNA repair synthesis, as evaluated by quantitative autoradiography, was present in thyreocytes exposed to DAA but absent after treatment with MDB, PTU, and THA. Consistent with their thyroid-specific carcinogenic activity, all the four chemicals, administered p.o. in rats in a single dose corresponding to 1/2 LD50, induced a statistically significant degree of DNA fragmentation in the thyroid, whereas any substantial evidence of DNA lesions was absent in liver, kidney, and lung, which, with the exception of liver tumors caused by THA, are not targets of the carcinogenic activity of the four test compounds. These findings indicate that the DNA damage observed in thyroid cells was consistent with the carcinogenicity of the four test compounds, and suggest that DAA, MDB, PTU, and THA might be carcinogenic to thyroid in humans.     

Metabolism of arsenic in primary cultures of human and rat hepatocytes.

The liver is considered a major site for methylation of inorganic arsenic (iAs). However, there is little data on the capacity of human liver to methylate iAs. This work examined the metabolism of arsenite (iAs(III)), arsenate (iAs(V)), methylarsine oxide (MAs(III)O), methylarsonic acid (MAs(V)), dimethylarsinous acid (DMAs(III)), and dimethylarsinic acid (DMAs(V)) in primary cultures of normal human hepatocytes. Primary rat hepatocytes were used as methylating controls. iAs(III) and MAs(III)O were metabolized more extensively than iAs(V) and MAs(V) by either cell type. Neither human nor rat hepatocytes metabolized DMAs(III) or DMAs(V). Methylation of iAs(III) by human hepatocytes yielded methylarsenic (MAs) and dimethylarsenic (DMAs) species; MAs(III)O was converted to DMAs. The total methylation yield (MAs and DMAs) increased over the range of 0.1 to 4 microM iAs(III). However, DMAs production was inhibited by iAs(III) in a concentration-dependent manner, and the DMAs/MAs ratio decreased. iAs(III) (10 and 20 microM) inhibited both methylation reactions. Inhibition of DMAs synthesis resulted in accumulation of iAs and MAs in human hepatocytes, suggesting that dimethylation is required for iAs clearance from cells. Methylation capacities of human hepatocytes obtained from four donors ranged from 3.1 to 35.7 pmol of iAs(III) per 10(6) cells per hour and were substantially lower than the methylation capacity of rat hepatocytes (387 pmol of iAs(III) per 10(6) cells per hour). The maximal methylation rates for either rat or human hepatocytes were attained between 0.4 and 4 microM iAs(III). In summary, (i) human hepatocytes methylate iAs, (ii) the capacities for iAs methylation vary among individuals and are saturable, and (iii) moderate concentrations of iAs inhibit DMAs synthesis, resulting in an accumulation of iAs and MAs in cells.     

Correlation between induction of DNA fragmentation and micronuclei formation in kidney cells from rats and humans and tissue-specific carcinogenic activity

Five chemicals, known to induce kidney tumors in rats, were assayed for their ability to induce DNA damage and formation of micronuclei in primary cultures of rat and human kidney cells and in the kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, and of micronuclei frequency were obtained in primary kidney cells from both rats and humans with the following concentrations of the five test compounds: lead acetate (not tested for micronuclei induction) and potassium bromate from 0.56 to 1.8 mM, phenacetin from 1 to 3.2 mM, and 1, 4-dichlorobenzene and nitrilotriacetic acid from 1.8 to 5.6 mM. In terms of DNA-damaging potency all the five chemicals were more active in rat than in human kidney cells, whereas the potencies in inducing micronuclei formation were similar in the two species with the exception of 1,4-dichlorobenzene, which was slightly more potent in human than in rat cells. Consistently with the results observed in vitro, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were detected in the kidney of rats given po a single (12 LD50) or three successive daily doses (13 LD50) of the five test compounds. 4, 4'-Methylenedianiline, a carcinogen which does not induce kidney tumors in rats, gave negative responses in both in vitro and in vivo assays. These findings give evidence that kidney carcinogens may be identified by short-term genotoxicity assays using as target kidney cells and show that the five chemicals tested produce in primary cultures of kidney cells from human donors effects similar to those observed in rats.     

Species differences in the induction of hepatocellular DNA synthesis by diethanolamine.

Diethanolamine increased the incidence and multiplicity of liver tumors in the mouse following chronic exposure. Diethanolamine is known to inhibit cellular choline uptake. Since choline deficiency produces tumors in rodents, diethanolamine, through choline depletion, may result in tumor development in rodents. The potential for diethanolamine to function through this mode of action in humans is not known. The present studies examined the effect of diethanolamine (0-500 mug/ml) and choline depletion on DNA synthesis and changes in expression of genes involved in cell growth pathways in primary cultures of mouse, rat, and human hepatocytes. In mouse and rat hepatocytes DNA synthesis was increased following treatment with 10 mug/ml diethanolamine and higher (3- to 4-fold over control). In contrast, diethanolamine failed to increase DNA synthesis in human hepatocytes. Incubation of hepatocytes in medium containing reduced choline (1/10 to 1/100 of normal medium; 0.898 to 0.0898 mg/l vs. 8.98 mg/l) increased DNA synthesis (1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively); however, choline depletion did not induce DNA synthesis in human hepatocytes. Mouse and rat hepatocytes incubated in medium supplemented with 2- to 50-fold excess choline reduced diethanolamine-induced DNA synthesis to control levels or below. Gene expression analysis of mouse and rat hepatocytes following diethanolamine treatment showed increases in genes associated with cell growth and decreases in expression of genes involved in apoptotic pathways. These results support the hypothesis that choline depletion is central to the mode of action for the induction of rodent hepatic neoplasia by diethanolamine. Furthermore, since diethanolamine treatment or choline depletion failed to induce DNA synthesis in human hepatocytes, these results suggest that humans may not be at risk from the carcinogenic effects of diethanolamine.     

Effects of bergamottin on human and monkey drug-metabolizing enzymes in primary cultured hepatocytes.

We investigated the effect of bergamottin, a major furanocoumarin in grapefruit juice, on phase I and phase II drug-metabolizing enzymes using cultured human and monkey hepatocytes. Both cultured systems were compared and evaluated for the direct effects of bergamottin as well as control treatments on liver enzymes. Treatment of hepatocytes with 0.1, 1, 5, and 10 microM bergamottin resulted in a concentration-dependent reduction in CYP3A4 activity (40-100%) in both human and monkey cells, as measured by testosterone 6 beta-hydroxylase activity. Bergamottin was potent at eliciting these inhibitory effects at both basal and induced states of CYP3A. Bergamottin (5 microM) completely inhibited alpha-naphthoflavone-induced ethoxyresorufin O-dealkylase (EROD) and methoxyresorufin O-dealkylase (MROD) activities in human hepatocytes and caused a 100% decrease in EROD activity in monkey hepatocytes. A 48-h exposure of cultured human hepatocytes to bergamottin resulted in increased levels of immunoreactive CYP3A4, CYP1A1, and CYP1A2 proteins, and CYP3A4, CYP1A1, CYP1A2, CYP2B6, and UDP-glucuronosyl transferase mRNAs. There was only a 20 to 30% reduction in glucuronidation and sulfation of 4-methylumbelliferone in human hepatocytes by 10 microM bergamottin and no effect on conjugation in the monkey hepatocytes. These results suggest that bergamottin causes both inhibition of CYP3A and CYP1A1/2 enzymatic activities and induction of correspondent proteins and mRNAs.     

Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse,rat, and human primary hepatocyte proliferation

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2′-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.     

Differences between human, rat and guinea pig hepatocyte cultures. A comparative study of their rates of beta-oxidation and esterification of palmitate and their sensitivity to R-etomoxir

Rat hepatocyte cultures have higher rates of beta-oxidation of palmitate and lower rates of esterification to glycerolipid than human or guinea pig hepatocytes. The R-enantiomer of etomoxir (sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate), a hypoglycaemic compound and inhibitor of carnitine palmitoyltransferase I, inhibited palmitate beta-oxidation in all three species, but the sensitivity to inhibition was highest in human hepatocytes and lowest in rat hepatocytes. The concentration causing half-maximal inhibition was approximately: 0.1 microM in human; 1 microM in guinea pig and 10 microM in rat hepatocytes. In human and in guinea pig hepatocytes the inhibition of beta-oxidation by R-etomoxir was associated with an increase in the esterification of palmitate but in rat hepatocytes R-etomoxir lowered the total rate of palmitate metabolism. The S-enantiomer of etomoxir had no significant effect on beta-oxidation or esterification of palmitate in any of the three species. It is concluded that there are significant differences between human, rat and guinea pig hepatocytes, not only in the relative partitioning of palmitate between beta-oxidation and esterification, but also in the sensitivity to an inhibitor of carnitine palmitoyltransferase I.     

Quinone-induced DNA single strand breaks in rat hepatocytes and human chronic myelogenous leukaemic K562 cells.

In rat hepatocytes exposed to the quinones menadione and 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) a decrease in NAD+ is observed. DNA damage and activation of poly(ADP-ribose)polymerase are often associated with a decrease in NAD+. Using rat hepatocytes and human myeloid leukaemic cells (K562), we examined the extent of DNA damage induced by these quinones at non-toxic concentrations, i.e. at concentrations at which the cells completely exclude the dye trypan blue. Both quinones caused significant DNA damage at very low concentrations (5-100 microM). With 2,3-diOME-1,4-NQ (15 microM) or menadione (15 microM) single strand breaks (SSB) were observed at very early time points (less than 5 min), reaching a maximum between 20 and 30 min. Most SSB were repaired within 45 min of the removal of the quinones. Whilst extensive repair was observed within 4 hr of the removal of 2,3-diOMe-1,4-NQ (15 microM), only partial repair was observed following exposure to menadione (15 microM). SSB induced by 2,3-diOMe-1,4-NQ (15 microM) were completely inhibited by the iron chelator 1,10-phenanthroline (25 microM), whereas in cells exposed to menadione (15 microM) they were only partially inhibited. Finally, although the membrane integrity of K562 cells was unaffected by exposure to high concentrations of both quinones (less than or equal to 400 microM), cytostasis was observed at much lower concentrations (50 microM). Our results demonstrate that at very low concentrations these quinones induce extensive DNA damage possibly caused by hydroxyl radicals. The DNA damage was accompanied by an early cytostasis but no loss of membrane integrity.     

Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells

Biomethylation is considered a major detoxification pathway for inorganic arsenicals (iAs). According to the postulated metabolic scheme, the methylation of iAs yields methylated metabolites in which arsenic is present in both pentavalent and trivalent forms. Pentavalent mono- and dimethylated arsenicals are less acutely toxic than iAs. However, little is known about the toxicity of trivalent methylated species. In the work reported here the toxicities of iAs and trivalent and pentavalent methylated arsenicals were examined in cultured human cells derived from tissues that are considered a major site for iAs methylation (liver) or targets for carcinogenic effects associated with exposure to iAs (skin, urinary bladder, and lung). To characterize the role of methylation in the protection against toxicity of arsenicals, the capacities of cells to produce methylated metabolites were also examined. In addition to human cells, primary rat hepatocytes were used as methylating controls. Among the arsenicals examined, trivalent monomethylated species were the most cytotoxic in all cell types. Trivalent dimethylated arsenicals were at least as cytotoxic as trivalent iAs (arsenite) for most cell types. Pentavalent arsenicals were significantly less cytotoxic than their trivalent analogs. Among the cell types examined, primary rat hepatocytes exhibited the greatest methylation capacity for iAs followed by primary human hepatocytes, epidermal keratinocytes, and bronchial epithelial cells. Cells derived from human bladder did not methylate iAs. There was no apparent correlation between susceptibility of cells to arsenic toxicity and their capacity to methylate iAs. These results suggest that (1) trivalent methylated arsenicals, intermediary products of arsenic methylation, may significantly contribute to the adverse effects associated with exposure to iAs, and (2) high methylation capacity does not protect cells from the acute toxicity of trivalent arsenicals.     

Assessment of induction of cytochrome P450 by NO-1886 (ibrolipim), a lipoprotein lipase-promoting agent, in primary cultures of human hepatocytes and in female rat liver

The mRNA levels of human cytochrome P450 (CYP)2Cs and CYP3As in primary cultures of freshly isolated human hepatocytes were assessed after exposure to NO-1886 and rifampicin, a typical inducer of CYP3As. mRNA levels were analyzed by real-time RT-PCR using an ABI PRISM 7700 Sequence Detector system. Exposure to NO-1886 for 24 hr at a concentration of less than 10 microM showed only a tendency to reduce or increase the expression levels of CYP2C8, CYP2C9, CYP2C19, CYP3A4, or CYP3A5 mRNA. A higher concentration (50 microM) of NO-1886 induced an increase in CYP2C8 mRNA and a decrease in CYP2C19 mRNA, and these changes continued after additional culture for 24 hr in fresh medium without NO-1886. The expression level of CYP3A4 mRNA after exposure to NO-1886 for 24 hr at 50 microM was about twice that in controls. Following additional culture for 24 hr in fresh medium without NO-1886, the expression of CYP3A4 mRNA was comparable to that in controls. On the other hand, the expression levels of CYP2C9 and CYP3A5 mRNA showed small and variable changes in each donor even at a high concentration (50 microM) of NO-1886. Furthermore, the pharmacokinetics of NO-1886 during repeated oral administration for 14 days was studied in female rats. The pharmacokinetic parameters of NO-1886 were nearly the same on days 1, 7, and 14 of repeated administration. The hepatic microsomal content of CYP isoforms was not affected by repeated administration for 7 days at a dose of 1 to 30 mg/kg in female rats, although the total CYP content was increased at a dose of 30 mg/kg. The expression levels of CYP1A2, CYP2B2, CYP2C12, and CYP2E1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2, 10, or 50 microM. The expression levels of CYP3A1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2 or 10 microM, but were increased, with large individual variation, by exposure at 50 microM. The mRNA expression levels in rat hepatocytes exposed to concentrations comparable to free plasma levels did not change significantly, which was consistent with the equivalence in the in vivo plasma concentrations observed on days 1 and 14 of repeated administration. These results suggest that repeated administration of NO-1886 at clinical doses does not significantly affect the expression levels of CYP isoforms in human liver, although the mRNA levels of the CYP isoforms involved in the metabolism of NO-1886 were increased by exposure to higher concentrations of NO-1886 in human hepatocytes in vitro.     

Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro

Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor β1 (TGFβ1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal β-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of β-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents. Received: 16 August 1999 / Accepted: 21 September 1999     

Effects of borneol on the level of DNA damage induced in primary rat hepatocytes and testicular cells by hydrogen peroxide

The aim of this paper was to evaluate genotoxic effects of borneol and its ability to change DNA-damaging effects of H₂O₂ in rat hepatocytes and testicular cells. Both in vitro and ex vivo approaches were used in the case of hepatocytes. Testicular cells were tested only ex vivo, i.e. shortly after isolation from rats supplemented by borneol. Cytotoxicity of borneol increased in in vitro conditions in a concentration-dependent manner and it was associated with DNA-damaging effects at toxic concentrations. While non-toxic concentrations of borneol applied in vitro protected cells against H₂O₂-induced DNA damage and interfered only partly with rejoining of H₂O₂-induced DNA strand breaks, cytotoxic concentrations of borneol manifested synergy with H₂O₂, i.e. enhanced DNA-damaging effects of H₂O₂. On the other side, borneol given to rats in drinking water decreased the level of DNA damage induced by H₂O₂ in both hepatocytes and testicular cells. Our results show that though at higher concentrations (2-h treatment with >2 mM borneol >0.3084 mg/ml) borneol acts cytotoxically and genotoxically on primary hepatocytes cultured in vitro, if given to rats during 7 days in a daily concentration of 17.14 or 34.28 mg/kg it reduces genotoxicity of H₂O₂ in both hepatocytes and testicular cells.     

Cytoprotection and iron mobilization in rat hepatocyte cultures by a new synthetic dihydroxamate chelator

The cytoprotection and iron mobilization effect of a new dihydroxamate chelator 1,1 bis [(11-N-hydroxy)-2,5,11-triaza-1,6,10-trioxo dodecanyl] ethane or KD was studied in primary rat hepatocyte cultures exposed to iron-citrate. Lactate dehydrogenase (LDH) release and malondialdehyde (MDA) production were measured as indexes of cytotoxicity. Cell viability was evaluated using the [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] (MTT) reduction test. To demonstrate that this chelator was able to decrease iron uptake or increase iron release from the hepatocytes, labelled cells were obtained by maintaining the cultures in the presence of 0.02 microM 55Fe-citrate. The efficacy of KD was compared to desferrioxamine B (DFO) at stoechiometry concentrations. After 24 h of exposure to 50 microM of iron-citrate, a significant release of LDH and MDA was observed. Cell viability was also significantly decreased. When 100 microM of KD were added at the same time as iron, LDH and MDA release was decreased and cell viability was improved. In the presence of the same chelator concentration, a net decrease of iron uptake by the cells was observed as attested by the low intracellular 55Fe level. Moreover, in the 55Fe loaded hepatocytes, the chelator increased the iron extracellular level indicating its iron release effect from the cells. In all tested experimental conditions, the efficacy of 100 microM of the dihydroxamate chelator KD was close to that of 50 microM of the trihydroxamate chelator DFO. In conclusion, KD is effective at a level comparable to DFO in protecting rat hepatocytes against the toxic effect of iron-citrate by decreasing the uptake of the metal and increasing its release from the cells. This synthetic compound appears to have some potential therapeutical interest and the results obtained encourage the synthesis of new hydroxamate ligands.     

Toxicity assessment of toxins T-514 and T-544 of buckthorn (Karwinskia humboldtiana) in primary skin and liver cell cultures.

The present study was undertaken to assess and compare the in vitro cytotoxicity of toxins T-514 and T-544 of buckthorn (Karwinskia humboldtiana) using primary cultures of rat hepatocytes and keratinocytes. Cell cultures were exposed to 6, 12, 25 and 50 microM toxins for 2-, 4-, 6- and 24-h periods. Cytotoxicity was determined by release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), in culture media, methylthiazoltetrazolium (MTT) reduction and neutral red (NR) uptake. An increase in LDH leakage was observed in liver cell cultures as early as 2 h with 50 microM T-544 and with 6 microM T-514 and T-544 at 6 h and 24 h, respectively. In the NR assay the toxicity was evident at 2 h with 12 microM T-514 and T-544 and with 6 microM concentrations of both toxins at 6 h. On the other hand, a decrease in MTT reduction was detected at 4 h with 50 microM concentrations of both toxins and with 25 microM T-544 and 12 microM T-514 at 6 h and 6 microM T-514 and T-544 at 24 h. Both toxins were shown to be highly hepatotoxic; T-514 was more toxic than T-544. In the skin cell cultures, the toxicity of the toxins was not as severe and was not expressed until 12 h of exposure.