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.     

Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity

Seven chemicals, six of which are known to induce epithelial neoplasms of the urinary bladder in rats, were assayed for their ability to induce DNA damage in primary cultures of rat and human cells from urinary bladder mucosa, and in urinary bladder, liver and kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, were obtained in cells from both rats and humans with the following concentrations of five test compounds: 2-naphthylamine and N-nitrosodi-n-butylamine 0.5 and 1 mM, phenacetin 2 and 4 mM, cyclophosphamide from 2 to 8 mM, and o-toluidine 16 and 32 mM. Nitrilotriacetic acid (1-4 mM), a rat bladder carcinogen, and 4-aminobiphenyl (0.125-0.5 mM), a bladder carcinogen in humans but not in rats, gave a weak positive response in rats cells and a more marked response in humans cells. In terms of DNA-damaging potency, 4-aminobiphenyl, cyclophosphamide, phenacetin and 4 nitrilotriacetic acid were more active in human than in rat cells, whereas the converse occurred with 2-naphthylamine. Consistently with the results observed in vitro statistically significant dose-dependent increases in the average frequency of DNA breaks were detected in the urinary bladder mucosa of rats given p.o. single doses corresponding to 14 and 12 LD50 of six of the seven test compounds; the only one which gave a substantially negative response was 4-aminobiphenyl. With the exception of N-nitrosodi-n-butylamine which caused DNA damage in liver and of phenacetin and nitrilotriacetic acid which caused damage in kidney in agreement with their tumorigenic activity, any substantial evidence of DNA lesions in these two organs was absent in rats treated with 12 LD50 of the other 4 test compounds. These findings give evidence that urinary bladder genotoxic carcinogens may be identified by the DNA damage/Comet assay using as targets cells of urinary bladder mucosa, and show that the effect may be quantitatively different in cells from rats and from human donors.     

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.     

DNA damage induced by 4,4'-methylenedianiline in primary cultures of hepatocytes and thyreocytes from rats and humans

4,4'-Methylenedianiline (MDA), an aromatic amine used in various industrial processes and previously found to induce tumor development in liver and thyroid of mice and rats, was evaluated for its DNA-damaging activity in primary cultures of hepatocytes and thyreocytes from rat and human donors. After exposure for 4 and 20 h to MDA concentrations ranging from 10 to 180 microM, a statistically significant increase in the frequency of DNA lesions was revealed by the Comet assay in primary hepatocytes and thyreocytes from donors of both species, the response being dose dependent up to 56-100 microM MDA. DNA fragmentation was more marked after 4 than after 20 h exposure in all four cell types. DNA was damaged to a lesser extent in human hepatocytes and thyreocytes than in corresponding rat cells and in both species in hepatocytes than in thyreocytes. In both rat and human hepatocytes a 20-h exposure to the same MDA concentrations elicited a modest amount of DNA repair synthesis, as evaluated by autoradiography. Evidence of a partial reduction of DNA damage, and therefore of only partial DNA repair, was observed in rat hepatocytes and in rat and human thyreocytes incubated for 16 h in MDA-free medium after a 4-h MDA treatment. A 4-h exposure to 56, 100, and 180 microM MDA did not induce DNA lesions in primary cultures of cells from three rat organs, kidney, urinary bladder mucosa, and brain, which are resistant to MDA carcinogenic activity. Under the same experimental conditions any evidence of DNA damage was absent in primary kidney and urinary bladder cells from human donors. Taken as a whole the results of this work indicate that MDA is specifically activated to DNA-damaging reactive species by hepatocytes and thyreocytes in both rats and humans and thus suggest that liver and thyroid might be the targets of the carcinogenic activity of MDA also in humans.     

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.     

Toxic activity of seventeen industrial solvents and halogenated compounds on human lymphocytes cultured in vitro

Seventeen chemicals (solvents, insecticides and intermediates in the production of textiles and resins) were tested in a short-term in vitro system with human lymphocytes to determine their toxic action. The parameters studied were the tritiated thymidine uptake and cell viability in cultures grown with or without a rat liver metabolizing system (S-9 mix). Data obtained showed that 1,3-dichlorobenzene, 1,2-dichlorobenzene, hexane, 1,2-diiodoethane, 1,4-dichlorobenzene, tetrachloroethylene, 2,3-dibromopropanol, chloromethyl methyl ether, 1,2- and 1,3-dibromopropane, in order, exerted the more toxic effects; ethyl acetate, cyclohexane, cyclohexanone and benzene showed lower toxic activity. The chemicals lost their toxic power in the presence of the metabolizing system with the exception of 1,2- and 1,3-dichlorobenzene which maintained in some degree their toxicity even in the presence of the S-9 mix. Only chloromethyl methyl ether elicited unscheduled DNA synthesis acting as DNA damaging agent.     

DNA damage in tissues of rat treated with potassium canrenoate

Potassium canrenoate (PC), a competitive aldosterone antagonist previously found to increase tumor incidence in rats and to produce genotoxic effects in in vitro systems, was examined in rats to acquire information on its genotoxic activity in vivo. Intragastric administration of 1/2 LD50 produced, as revealed by the Comet assay, a modest but statistically significant increase in the frequency of DNA lesions in liver but not in thyroid and bone marrow of male rats, and in thyroid and bone marrow but not in liver of female rats. In contrast with the frankly positive responses observed in primary cultures of rat hepatocytes (Martelli et al., Mutagenesis 14 (1999) 463-472) any evidence of DNA repair and micronuclei formation was absent in liver of rats treated with 1/2 LD50, and initiation of enzyme-altered liver preneoplastic lesions did not occur in the liver of rats given 100 mg/kg PC once a week for 6 successive weeks. A high and dose-dependent frequency of DNA lesions was found to occur in testes and ovaries of rats given single doses ranging from 1/8 to 1/2 LD50.     

Assessment of chemically induced DNA repair in primary cultures of human bronchial epithelial cells

A procedure has been developed to assess chemically induced DNA repair in freshly isolated, primary cultures of human bronchial epithelial (HBE) cells. HBE cells were isolated from eight samples of autopsy material or surgical specimens and incubated with test chemicals and [3H]thymidine. Viability as measured by trypan blue exclusion averaged 90%. Chemically induced DNA repair was assessed as unscheduled DNA synthesis (UDS) by quantitative autoradiography. The direct-acting agent methyl methanesulfonate induced DNA repair in HBE cells in all eight cases studied, indicating that the cultures were viable and capable of DNA repair in response to DNA damage. Benzo(a)pyrene induced DNA repair in all cultures whereas dimethylnitrosamine failed to induce UDS in any culture, suggesting an organ-specific pattern of metabolic activation. 1,6-Dinitropyrene was positive in cultures prepared from autopsy material but negative in cultures prepared from surgical specimens. Formaldehyde did not induce UDS in any sample examined. This system may be useful in assessing the genotoxic potential of environmental chemicals in human bronchial epithelial cells, give an indication of interindividual variability, and provide valuable information for comparison to proposed animal models for the human bronchus.     

Human hepatocytes in genotoxicity assays

Human hepatocyte primary cultures, because of their comprehensive biotransformation capability, represent an experimental model particularly useful to gain direct information on the genotoxic risk of chemicals to humans. For this purpose they have been used in the last few years either as target cells to evaluate the induction of DNA damage and/or DNA repair synthesis, or as metabolic activation system in mutagenicity assays. The number of compounds so far tested is rather limited, and for the large majority of them the assays have been performed only on hepatocytes from a few donors. A comparison with the data obtained in rat hepatocytes indicates that quantitative differences in the genotoxic effects induced in cultures derived from different donors of the same species are usually greater than interspecies differences. However, the results.provided by some chemicals suggest the possibility that in certain cases rat hepatocytes might be inappropriate predictors of the genotoxic hazard for humans.     

A comparative study of chemically induced DNA damage in isolated human and rat testicular cells

Testicular cells prepared from human organ transplant donors or from Wistar rats were used to compare 15 known reproductive toxicants with respect to their ability to induce DNA damage, measured as single-strand DNA breaks and alkali labile sites (ssDNA breaks) with alkaline filter elution. The compounds tested included various categories of chemicals (i.e., pesticides, industrial chemicals, cytostatics, and mycotoxins) most of which are directly acting genotoxicants (i.e., reacting with DNA either spontaneously or via metabolic activation). In addition, a few indirect genotoxic and nongenotoxic reproductive toxicants were included. Six of the chemicals induced no significant levels of ssDNA breaks in human and rat testicular cells: methoxychlor (10 to 100 μM, human and rat), benomyl (10 to 100 μM, human and rat), thiotepa (10 to 1000 μM, human and rat), cisplatin (30 to 1000 μM, human; 100 to 1000 μM, rat), Cd²⁺ (30 to 1000 μM, human; 100 to 1000 μM, rat), and acrylonitrile (30 to 1000 μM, human; 30 to 300 μM, rat). Four chemicals induced significant levels of ssDNA breaks in testicular cells from both species: styrene oxide (≥ 100 μM, rat and human), 1,2-dibromoethane (EDB) (≥ 100 μM, rat; 1000 μM human), thiram (≥ 30 μM, rat; ≥ 100 μM, human), and chlordecone (300 μM, rat; ≥ 300 μM, human). Finally, five chemicals induced ssDNA breaks in one of the two species. Four chemicals induced significant ssDNA breaks in rat testicular cells only: 1,2-dibromo-3-chloropropane (DBCP) (≥ 10 μM), 1,3-dinitrobenzene (1,3-DNB) (≥ 300 μM), Cr⁶⁺ (1000 μM), and aflatoxin B₁ (≥ 100 μM), the last two of these produced only a minor positive response. One chemical, acrylamide, induced a marginal increase in ssDNA breaks in human at 1000 μM, but not in rat testicular cells. Although based on a limited number of donors, the data indicate a close correlation between the induction of DNA damage in human and rat testicular cells in vitro. For some chemicals, however, there appears to be differences in the susceptibility to chemically induced ssDNA breaks of isolated testicular cells from the two species. The data indicate that the parallel use of human and rat testicular cells provides a valuable tool in the assessment of human testicular toxicity.     

Effect of Rodent Hepatocarcinogenic Peroxisome Proliferators on Fatty Acyl-CoA Oxidase, DNA Synthesis, and Apoptosis in Cultured Human and Rat Hepatocytes

The effects of the rodent hepatocarcinogens clofibric acid and ciprofibrate on the activity of the peroxisomal fatty acyl-CoA oxidase, DNA synthesis, and apoptosis were compared in cultured rat and human hepatocytes. Rat hepatocytes expressed a 10-fold greater level of the peroxisomal fatty acyl-CoA oxidase compared to human hepatocytes. At the highest concentration (1.0 mM), both drugs induced a two- to threefold increase in this enzyme activity in both rat and human hepatocytes. Ciprofibrate (0.1 and 0.2 mM) caused a twofold increase in DNA synthesis in rat hepatocytes, whereas clofibric acid had no effect on DNA synthesis in these cells. In contrast, increasing concentrations of both clofibric acid and ciprofibrate produced inhibition of DNA synthesis in human hepatocytes. By using the terminal transferase dUTP–biotin nick end labeling technique, it was observed that 0.1 and 0.2 mM clofibric acid and ciprofibrate suppressed transforming growth factor-β (TGFβ)-induced apoptosis by 50% in rat hepatocytes, but they had no effect on TGFβ-induced apoptosis in human hepatocytes. Although clofibric acid and ciprofibrate diminished TGFβ-induced apoptosis, they had no effect on the basal apoptotic levels in the rat hepatocyte cultures. However, both drugs significantly increased the percent of apoptotic cells in the human hepatocyte cultures. It is concluded that primary rat and human hepatocyte cultures respond differently to peroxisome proliferators. The differences in effects on DNA synthesis and apoptosis support the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatocarcinogenesis.     

Aroclor 1254 increases the genotoxicity of several carcinogens to liver primary cell cultures

The genotoxicity of both direct-acting and precarcinogenic chemicals was evaluated in liver primary cell cultures (LPCC) from untreated and Aroclor 1254 (Ar) pretreated rats. Hepatocytes were isolated from partially hepatectomized rats and their DNA was labeled in vitro with [3H] dThd; the molecular weight of single-stranded DNA was determined by alkaline sucrose sedimentation. Two parameters of DNA damage were defined: the mean effective dose (ED50), i.e., the carcinogen concentration that decreased the DNA molecular weight to half the original, and the DNA breaking potency (DBP), i.e., the number of breaks per DNA molecule produced by 2 h exposure to 1 mM concentration of the chemical. Two hours exposure of LPCC from untreated rats to the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (6.8-340 microM) and to the precarcinogens benzo[a]pyrene (BaP) (0.05-0.33 mM) and dimethylnitrosamine (DMN) (0.45-16 mM) produced a concentration-dependent decrease in the molecular weight of DNA. Pretreatment of rats with Ar decreased significantly the sedimentation velocity of DNA and increased five, three, and two times the DBP of MNNG, BaP, and DMN, respectively. These results show that Ar-pretreatment of rats increases the genotoxicity of both direct-acting and precarcinogenic chemicals and suggest that Ar might increase the genotoxicity of chemical carcinogens perhaps by enhancing their metabolic activation, by producing direct genotoxic effects, or both. Our results also emphasize the carcinogenic risk that the environmental pollution by polychlorinated biphenyls might represent to humans.     

Cytotoxicity of trichloroethylene and S-(1, 2-dichlorovinyl)-L-cysteine in primary cultures of rat renal proximal tubular and distal tubular cells

Activities of several glutathione-dependent enzymes, expression of cytochrome P450 isoenzymes, and time- and concentration-dependent cytotoxicity of trichloroethylene (TRI) and S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) were evaluated in primary cultures of proximal tubular (PT) and distal tubular (DT) cells from rat kidney. These cells exhibited cytokeratin staining and maintained activities of all glutathione-dependent enzymes measured. Of the cytochrome P450 isoenzymes studied, only CYP4A expression was detected. CYP4A mRNA and protein expression were higher in primary cultures of DT cells than in PT cells and were increased in DT cells by ciprofibrate treatment. Incubation of cells for 6 h with concentrations of TRI as high as 10 mM resulted in minimal cytotoxicity, as determined by release of lactate dehydrogenase (LDH). In contrast, marked cytotoxicity resulted from incubation of PT or DT cells with DCVC. Addition to cultures of TRI (2-10 mM) for 24 or 72 h resulted in modest, but significant time- and concentration-dependent increases in LDH release. Treatment of cells with DCVC (0.1-1 mM) for 24 h caused significant increases in LDH release and alterations in cellular protein and DNA content. Finally, exposure of primary cultures to TRI or DCVC for 72 h followed by 3 h of recovery caused a slight increase in the expression of vimentin, consistent with cellular regeneration. These studies demonstrate the utility of the primary renal cell cultures for the study of CYP4A expression and mechanisms of TRI-induced cellular injury.     

UDS induction by an array of standard carcinogens in human and rodent hepatocytes: effect of cryopreservation

The UDS induction assay with primary hepatocytes as the target cells is a determinative assay for chemical carcinogens. This assay is, however, limited to the availability of freshly prepared liver cells. A cryopreservation technique for liver cells has recently been described. Frozen cells have been shown to retain a variety of enzyme activities essential for xenobiotic metabolism after being thawed. In the present investigation, 19 direct or indirect-acting carcinogens were tested with respect to their capacity to induce DNA repair in primary as well as cryopreserved human and rat hepatocytes. Cryopreserved cells yielded results that were essentially indistinguishable from fresh cells. Only marginal differences were observed between hepatocytes of rat or human origin. These results demonstrate the suitability of cryopreserved hepatocytes as indicator cells for the study of UDS induction to discover possible carcinogenicity in chemicals.     

Evaluation of flutamide genotoxicity in rats and in primary human hepatocytes

Flutamide, an effective competitive inhibitor of the androgen receptor used orally for palliative treatment of prostatic carcinoma and regulation of prostatic hyperplasia was evaluated for its genotoxic effects in the intact rat and in primary cultures of human hepatocytes. Negative responses were obtained in all the in vivo assays as well as in the in vitro assay. In rats given a single oral dose of 500 mg/kg flutamide, fragmentation and repair of liver DNA were absent, and no increase was observed in the frequency of micronucleated hepatocytes. In the liver of rats given flutamide as initiating agent at the dose of 500 mg/kg/week for 6 successive weeks, gamma-glutamyltraspeptidase-positive foci were detected only in 3 of 10 rats. There was no evidence of a promoting effect on the development of aberrant crypt foci in rats given 100 mg/kg flutamide on alternate days for 8 successive weeks. In primary cultures of human hepatocytes from one male and one female donor DNA fragmentation as measured by the Comet assays, and DNA repair synthesis as revealed by quantitative autoradiography, were absent after a 20 hr exposure to flutamide concentrations ranging from 18 to 56 microM. Taken as a whole, our results seem to indicate that flutamide is a non-genotoxic drug.     

A comparative study of chemically induced DNA damage in isolated nasal mucosa cells of humans and rats assessed by the alkaline comet assay

Single-cell microgel electrophoresis (comet) assay was used to study genotoxic effects in human nasal mucosa cells and rat nasal and ethmoidal mucosa cells in vitro. Human cells were obtained from tissue samples of 10 patients (3 females/7 males), who underwent surgery (conchotomy) for treatment of nasal airway obstruction. Rat nasal mucosa cells were derived from male Sprague-Dawley rats. Cells were exposed for 1 h to either N-nitrosodiethanolamine (NDELA), epichlorohydrin (EPI), 1,2-epoxybutane (EPB), ethylene dibromide (EDB), or 1,2-dibromo-3-chloropropane (DBCP). Dimethyl sulfoxide (DMSO) was used as negative control. Alkaline comet assay was performed according to a standard protocol and DNA damage was quantified as Olive tail moment using image analysis system. All test substances induced an increase in DNA damage in human and rat cells. The absolute amount of DNA damage in rat nasal mucosa cells was usually higher than in ethmoidal mucosa cells. Human nasal mucosa cells were found to be less sensitive than rat mucosa cells to the genotoxic activities of DBCP (lowest effective concentration in human cells [LEC(human)]: 1.5, in rat cells [LEC(rat)]: 0.01 mM) and NDELA (LEC(human): 25, LEC(rat): 12.5 mM), whereas EPB-treated cells were almost equal (LEC(human) and LEC(rat) 0.78 mM). NDELA induced a marked concomitant cytotoxicity. For EPI (LEC(human) and LEC(rat): 0.097 mM) and EDB (LEC(human): 0.195, LEC(rat): 0.048 mM), pronounced interindividual differences were observed in human samples.     

Preferential induction of CYP1A1 and CYP1B1 in CCSP-positive cells.

Both benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands of aryl hydrocarbon receptors (AhR). Although animal studies indicate that both compounds induce pathological changes in the peripheral lung, the specific cell type involved remains unclear. Clara cells, expressing Clara cell specific protein (CCSP) and abundant in cytochrome P450, are nonciliated bronchiolar epithelial cells in the peripheral lung. Here we explore the hypothesis that CCSP-positive Clara cells are highly responsive to AhR ligands and are the primary cell type involved in BaP- and TCDD-induced toxicities. The responsiveness to AhR ligands was evaluated by measuring the respective mRNA and protein levels of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) using real-time RT-PCR and immunocytochemistry assays. Two in vitro models were used: primary cultures of human small airway epithelial (SAE) cells and rat lung slice cultures. In the presence of calcium, human SAE cells differentiated into CCSP-positive cells. BaP- and TCDD-induced mRNA and protein levels of CYP1A1 and CYP1B1 levels were significantly elevated in CCSP-positive cell cultures. Similarly, AhR mRNA and protein levels were increased in CCSP-positive cell cultures, as determined by real-time RT-PCR and Western blot analysis. When rat lung slice cultures were treated with BaP or TCDD for 24 h, CYP1A1 and CYP1B1 proteins were strongly induced in Clara cells. These results indicate that, in the peripheral lung of both rats and humans, CCSP-positive cells (Clara cells) may be more sensitive to AhR ligands than other cell types.     

Comparison of TCDD and PCB CYP1A induction sensitivities in fresh hepatocytes from human donors, sprague-dawley rats, and rhesus monkeys and HepG2 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals induce cytochrome P450 1A (CYP1A) gene expression and, at sufficient exposures, cause toxicity. Human health risks from such exposures are typically estimated from animal studies. We tested whether animal models predict human sensitivity by characterizing CYP1A gene expression in cultures of fresh hepatocytes from human donors, rats, and rhesus monkeys and HepG2 human hepatoma cells. We exposed the cells to three aryl hydrocarbon receptor (AhR) ligands of current environmental interest and measured 7-ethoxyresorufin-O-deethylase (EROD) activity and concentrations of CYP1A1 and CYP1A2 mRNA. We found that human cells are about 10-1000 times less sensitive to TCDD, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and Aroclor 1254 than rat and monkey cells, that relative potencies among these chemicals are different across species, and that gene expression thresholds exist for these chemicals. Newly calculated rat-human interspecies relative potency factors for PCB 126 were more than 100 times lower than the current rodent-derived value. We propose that human-derived values be used to improve the accuracy of estimates of human health risks.     

2-Bromopropane induces DNA damage,impairs functional antioxidant cellular defenses,and enhances the lipid peroxidation process in primary cultures of rat Leydig cells

Utilization of highly enriched preparations of steroidogenic Leydig cells has proven invaluable for studying the direct effects of various hormones and agents on Leydig cell function in vitro. It is widely reported that male reproductive organs are particularly susceptible to the deleterious effects of reactive oxygen species (ROS) and lipid peroxidation, which ultimately lead to impaired fertility. The purpose of the study was to examine the potential of 2-bromopropane (2-BP) to induce oxidative stress and antioxidant function in primary cultures of rat Leydig cells. Leydig cells were isolated from the testes of Sprague-Dawley rats. The purity of Leydig cells was determined to be 94.6% and the cells maintained their testosterone secreting capabilities for 48 h. Fresh medium containing 2-BP (1.00, 0.10, 0.01 mM, and vehicle control) and 1 U human chorionic gonadotropin (hCG) were added in the cell culture. Superoxide dismutase (SOD) activity, malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) were analyzed in the medium of each well by biochemical methods. Additionally, DNA damage was examined using the Comet assay. The proportion of cells with undamaged DNA was decreased significantly and those with different grades of damaged DNA were increased significantly in the cells exposed to 2-BP. The level of MDA and GSH-PX activity increased significantly in the cell groups exposed to 0.10 and 1.00 mM 2-BP, whereas, SOD activity decreased considerably in these two groups of cells when compared to the control. The data indicate that 2-BP induces DNA damage, impairs functional antioxidant cellular defenses, and enhances the lipid peroxidation in cultured Leydig cells. These effects may be responsible for the testicular toxicity noted in laboratory animals and humans.     

Editorial

Mass cultures of primary rat kidney cells were exposed briefly to aqueous solutions of 20 chemicals and their subsequent growth rate and mitotic activity measured. Proximate carcinogens, and some chemicals with a defined inhibitory action biochemically, depressed the growth and division of the cultures. Non-carcinogenic compounds and precarcinogens did not interfere with the subsequent growth rate and mitotic activity of the cultures. It is suggested that the possession of growth inhibitory properties could give an indication of the carcinogenic activity of a chemical on a short-term basis.