Cryopreservation and long-term storage of primary rat hepatocytes: Effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P45O-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at –196°C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylaminofluorene, 7,12-dimethyEbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.Abbreviations 2-AAF 2-acetylaminofluorene - DDH₂O distilled deionized water - DMBA 7,12-dimethyIbenz[a]anthracene - DMN dimethylnitrosamine - DMNA dimethylnitrosamine-N-demethylase - DMSO dimethyl sulfoxide - EROD 7-ethoxyresorufin-O-deethylase - F344 Fischer 344 - FBS fetal bovine serum - %IR percentage of cells in repair - LN₂ liquid nitrogen - LSD least significant difference - CG cytoplasmic grains - NNG net nuclear grains - SD Sprague-Dawley - UDS unscheduled DNA synthesis - WE Williams' Medium E     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

DNA damage and repair in gamma-glutamyltranspeptidase-positive and negative hepatocytes in primary culture from carcinogen-treated rats.

Chemically induced DNA fragmentation and unscheduled DNA synthesis were determined in gamma-glutamyltranspeptidase (GGT)-positive and GGT-negative hepatocytes isolated from rat livers subjected to a multistage hepatocarcinogenesis regimen (Solt-Farber), which included 0.05% phenobarbital promotion for 6 weeks (early) or 6 months (late). The results indicated that there was DNA damage in untreated GGT-positive and GGT-negative hepatocytes with either period of promotion compared with normal hepatocytes; however, no statistical difference could be seen between GGT-positive and GGT-negative hepatocytes. DNA damage induced in vitro by the activation-dependent carcinogen dimethylnitrosamine was much less in GGT-positive hepatocytes than in GGT-negative hepatocytes or normal hepatocytes. No significant difference in DNA damage was seen in both GGT-positive and GGT-negative cell populations following treatment with the activation-independent carcinogen ethylnitrosourea (ENU), although DNA damage of GGT-positive hepatocytes was less than that of normal hepatocytes. The background of unscheduled DNA synthesis in both GGT-positive and GGT-negative hepatocytes at either time of promotion was higher than that of normal hepatocytes. The capacity for DNA repair in GGT-positive hepatocytes appeared to be lower than that in GGT-negative hepatocytes. GGT-negative hepatocytes exhibited a lower capacity for DNA repair than that of normal hepatocytes in terms of the rate of unscheduled DNA synthesis elicited by dimethylnitrosamine and ethylnitrosourea in vitro.     

G2 sub-population in mouse liver induced into mitosis by lead acetate

A single intracardiac dose of lead acetate (40 microgram lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. The prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G2 phase. The injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. The incremental labeled cells may have originated from a small fraction of the G1 population that was ready to enter the S phase without the usual pre-synthetic delay.     

Fluorescence-activated sorting of rat hepatocytes based on their mixed function oxidase activities towards diethoxyfluorescein.

The formation of ethoxyfluorescein and fluorescein from diethoxyfluorescein by isolated rat hepatocytes has been used as a basis for separating such cells dependent on their mixed function oxidase activities by fluorescence-activated flow cytometry. Five equal fractions defined by computer-generated regions were isolated. Non-viable cells with low fluorescence (region 1) represented 10-15% of the population, while the remainder with higher mixed function oxidase activities (regions 2-5), were greater than 95% viable by Trypan Blue exclusion. In region 1, 30% of the viable cells were binucleate, 67% diploid while in region 5, 13% were binucleate and 69% tetraploid. At 3 h after sorting, following attachment to glass coverslips, exposure of cells to methyl methanesulphonate, retrorsine or norethindrone resulted in unscheduled DNA synthesis which was 2-fold higher in the tetraploid-rich region 5, while aflatoxin B1, benzo[a]pyrene or 2-acetylaminofluorene caused a 5-fold increase in unscheduled DNA synthesis in these cells, relative to the diploid-rich hepatocytes in region 2.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells.UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle.Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine.The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

G2 Sub-Population In Mouse Liver Induced Into Mitosis By Lead Acetate

A single intracardiac dose of lead acetate (40 μ lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. the prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G₂ phase. the injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. the incremental labeled cells may have originated from a small fraction of the G₁ population that was ready to enter the S phase without the usual pre-synthetic delay.     

Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.     

A sensitive method to measure physical and chemical carcinogen-induced "unscheduled DNA synthesis" in rapidly dividing eukaryotic cells

A sensitive assay for quantitating ‘unscheduled DNA synthesis’ (repair synthesis) in transformed human amnion (AV₃) cells has been developed. The combined use of hydroxyurea and arginine-deficient culture medium enabled the detection of 10–20 fold increases in ‘unscheduled DNA synthesis’ after treatment with N-acetoxy-2-acetylaminofluorene or ultraviolet light. The technique allows the detection of ‘DNA repair synthesis’ following treatment with extremely low doses of mutagens and carcinogens.     

alpha 1-Fetoprotein production during the hepatocyte growth cycle of developing rat liver.

The relation of AFP production to DNA synthesis was investigated in newborn rat liver and in primary cultures of fetal rat hepatocytes, by combining immunoperoxidase AFP localization and autoradiography after ³H-thymidine labelling. The vast majority of AFP-positive hepatocytes did not incorporate ³H-thymidine after ≤4-h isotope pulses, suggesting that in the developing liver, essentially no production of AFP occurs in S, G₂ or M phases of the hepatocyte cell life cycle. Serial or continuous thymidine labelling experiments further indicated that post-mitotic hepatocytes constitute a sizable fraction of AFP-producing cells.     

The effect of aging and dietary restriction on DNA repair

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.     

Differential sensitivity of human hepatoma cell line and primary rat hepatocyte culture to benzo(a)pyrene-induced unscheduled DNA synthesis and adduct formation.

We studied the genotoxic potential of a carcinogen in the human hepatoma cell line, HepG2 and in primary rat hepatocyte culture. HepG2 is a well differentiated human hepatoblastoma cell line with biotransforming capacity. Rat hepatocytes were obtained by the standard two-step in situ perfusion technique. Following benzo(a)pyrene treatment, both HepG2 and primary rat hepatocyte culture showed unscheduled DNA synthesis with different sensitivity. In ³²P-postlabelling analysis, the chromatogram revealed quantitative and qualitative differences between HepG2 and primary rat hepatocyte cultures when treated with 10 μM benzo(a)pyrene for 18 hr. The results have demonstrated that the HepG2 cell line may be used in addition to primary rat hepatocytes in risk assessment for detection of environmental carcinogens.     

The induction of DNA-strand breaks and unscheduled DNA synthesis in F-344 rat hepatocytes following in vivo administration of caprolactam or benzoin

Benzoin (ZOIN) and caprolactam (CAP) were administered by gavage to Fischer 344 rats at a dose of 750 mg/kg and the hepatocytes isolated 12, 24 or 48 h after treatment. The isolated hepatocytes were subsequently examined for the induction of DNA-strand breaks (SB) and unscheduled DNA synthesis (UDS). Neither ZOIN nor CAP induced SB or UDS in hepatocytes, however ZOIN did induce an increase in the fraction of cells in S-phase 24 h after treatment. These results correlate well with the observed lack of carcinogenicity of these compounds.     

Evidence that DNA repair may not be modified by age or chronic caloric restriction

Pretreatment of animals with mixed-function oxidase inducers has been shown to increase the metabolic activation capacity of isolated hepatocytes resulting in an apparent increase in DNA repair. We recently reported decreases in chemically-induced DNA repair, measured as unscheduled DNA synthesis (UDS), in hepatocyte cultures isolated from aging ad libitum (AL) and caloric restricted (CR) diet-fed animals. In the present study, we evaluated the effects of pretreatment with Aroclor 1254 (ARO) on the genotoxicity of 4 carcinogens, from different chemical classes, in primary hepatocytes isolated from male Fischer 344 rats. ARO-induced old AL- and CR-derived cultures, treated with 2-acetylaminofluorene (2-AAF), aflatoxin B₁ (AFB₁), 7,12-dimethylbenz[a]anthracene (DMBA), and dimethylnitrosamine (DMN), exhibited significant induction-related increases in DNA repair in comparison to uninduced old AL and CR animals. These data indicate that the constitutive levels of specific cytochrome P450 decline with age and chronic caloric restriction, while the ability to respond to exogenous inducers is retained, and suggest that DNA repair may not be modified with age or diet restriction.     

Testing of hydralazine in in vivo-in vitro hepatocyte assays for UDS and stimulation of replicative DNA synthesis

The vasodilator hydralazine was tested for induction of DNA-repair synthesis and stimulation of replicative DNA synthesis in rat hepatocytes after administration in vivo, either once or repetitively. No increase in unscheduled or replicative DNA synthesis was observed. By contrast, positive controls clearly induced DNA-repair synthesis, either after a single treatment (4-aminobiphenyl, dimethylnitrosamine and methyl methanesulphonate) or after repetitive treatment (benzo[a]pyrene), or stimulated replicative DNA synthesis (carbon tetrachloride and dimethylnitrosamine). Thus, hydralazine displayed no genotoxic and no tumour-promoting activity in these in vivo-in vitro test systems.     

N-Nitrosomorpholine induced alterations of unscheduled DNA synthesis, mitochondrial DNA synthesis and cell proliferation in different cell types of liver, kidney, and urogenital organs in the rat

In order to measure rates of unscheduled DNA synthesis (UDS), mitochondrial DNA synthesis, and cell proliferation, i.e. factors relevant in the early phase of carcinogenesis, young rats received by gavage 200 mg/kg N-nitrosomorpholine (NNM) or vehicle (distilled water), and were injected with 3H-thymidine 24 h later. Autoradiographs from liver, kidney, urethra, prostate, seminal vesicle, and ductus deferens were prepared from deparaffinized sections, using a 250-day exposure time. In the liver, UDS was at least doubled in 2n and 4n hepatocytes. Approximately 3% of these hepatocytes exhibited a fourfold increase in UDS. Such strongly labeled cells were only observed in the liver following NNM exposure. With the exception of renal epithelial cells of the proximal tubule, UDS in epithelial cells of bladder, urethra, ductus deferens, seminal vesicle and prostate was decreased in NNM-exposed rats. Mitochondrial DNA synthesis and cell proliferation were significantly increased only in hepatocytes, and were decreased in all other monitored organs in NNM-exposed rats. The strongly increased UDS and more moderately increased mitochondrial DNA synthesis in a subgroup of hepatocytes suggest that possibly some unrepaired damage persists in the DNA of these cells. The latter cells may be the precursors of so-called foci of hepatocellular alteration, which appear later during the process of carcinogenesis. The increased UDS but decreased rate of proliferation in the renal proximal tubule cells might be related to renal carcinogenesis which is observed in NNM-exposed rats after a long latency period.     

Unscheduled DNA synthesis in rat tracheal epithelial cells, hepatocytes and spermatocytes following exposure to methyl chloride in vitro and in vivo

Measurement of DNA repair as unscheduled DNA synthesis (UDS) in vitro following exposure in vivo in multiple tissues from the same treated animal can provide valuable information relating to the tissue- and organ-specificity of chemically induced DNA damage. UDS was evaluated in primary cultures of rat tracheal epithelial cells, hepatocytes and pachytene spermatocytes after exposure in vitro to methyl chloride (MeCl), and after isolation from the same treated animal following inhalation exposure in vivo. Concentrations of 1-10% MeCl in vitro induced UDS in hepatocytes and spermatocytes, but not in tracheal epithelial cells. Inhalation exposure to MeCl in vivo (3000-3500 ppm 6 h/day for 5 successive days) failed to induce DNA repair in any cell type. In vivo exposure to 15 000 ppm MeCl for 3 h also failed to induce UDS in tracheal epithelial cells and spermatocytes, but did cause a marginal increase in UDS in hepatocytes. Thus, MeCl appears to be a weak, direct-acting genotoxicant. While activity could be measured in hepatocytes and spermatocytes directly in vitro, only extremely high concentrations of MeCl elicited a response in the whole animal, and then only in hepatocytes.     

Individual variability of pathological parameters in chemically induced rat colon tumors.

The development of tumorigenic conditions in the carcinogen-exposed rat colon was studied using selected morphological, histochemical, immunohistochemical and biochemical methods of analysis. Rats were treated with two carcinogens: 1,2-dimethylhydrazine and N-methyl-N'-nitro-N-nitrosoguanidine alone or with deoxycholic acid as a tumor promoter. It was found that 3 months after treatment of animals with the carcinogens the following changes were developed in colonic tissue: infiltration of lymphocytes in the mucous membrane, high increase in mitotic index among epithelial cells, negative reactions of colonic cells for neutral mucopolysaccharides and sulfomucins and positive reactions to carboxyl groups, nonsulfated acid mucosubstances and tissue polypeptide antigens. An increase in the activity of ornithine decarboxylase in colonic tissue was developed within the same time period and has been seen only in those tissues which were characterized by the development of precancerous conditions. Individual variations were observed in the manifestation of the studied parameters in rat neoplastic colonic tissues. It is suggested that these differences reflect an individual sensitivity of animals to carcinogens and the magnitude of the dysplastic processes induced in the colon.     

N-nitrosodiethylamine genotoxicity evaluation: a cytochrome P450 induction study in rat hepatocytes

In rats, N-nitrosodiethylamine (NDEA) induces tumors mainly in the liver. This could be because various enzymes are responsible for the metabolic activation of NDEA, besides the hepatic NDEA metabolizing enzyme, CYP2E1. We examined NDEA genotoxicity and cytotoxicity in primary cultures of female rat hepatocytes; we also looked at how it affected CYP mRNA expression. Single incubation with 0.9% NaCl resulted in a mean of 0.2% apoptotic cells, which doubled with 105 μg NDEA/mL. The frequency of necrosis with NDEA treatment was also doubled. Besides the cytotoxic effects, there was also a 4-fold decrease in mitotic index and a 3-fold decrease in the percentage of cells with micronuclei. A significant increase in micronucleus cells when hepatocytes were incubated with 2.1 μg NDEA/mL suggests that DNA repair was inactive. The chromosomal aberration evaluation revealed a discrete dose-response curve. Treatment with NDEA induced increases in CYP mRNA: CYP2B2 (1.8 times) and CYP2E1 (1.6 times) with non-cytotoxic NDEA concentrations (0.21-21 μg/mL). CYP2B1 mRNA levels decreased at 0.21 μg NDEA/mL (2.5-fold), while CYP4A3 mRNA decreased 1.3-fold. NDEA treatment at 2.1 μg/ mL induced a 1.9-fold increase in CYP3A1 mRNA. Understanding the cumulative effects in target cells during precarcinogenesis is crucial to understanding the mode of action of potential carcinogens and in order to develop comprehensive chemical toxicity profiles.