Frequency Of DNA Breaks Research Articles

Circadian control of cisplatin-DNA adduct repair and apoptosis in culture cells.

Cisplatin, a widely prescribed chemotherapeutic agent for treating solid tumors, induces DNA adducts and activates cellular defense mechanisms, including DNA repair, cell cycle checkpoint control, and apoptosis. Considering the circadian rhythmicity displayed by most chemotherapeutic agents and their varying therapeutic efficacy based on treatment timing, our study aimed to investigate whether the circadian clock system influences the DNA damage responses triggered by cisplatin in synchronized cells. We examined the DNA damage responses in circadian-synchronized wild-type mouse embryonic fibroblasts (WT-MEF; clock-proficient cells), cryptochrome1 and 2 double knock-out MEF (CRYDKO; clock-deficient cells), and mouse hepatocarcinoma Hepa1c1c7 cells. Varying the treatment time resulted in a significant difference in the rate of platinum-DNA adduct removal specifically in circadian-synchronized WT-MEF, while CRYDKO did not exhibit such variation. Moreover, diurnal variation in other DNA damage responses, such as cell cycle checkpoint activity indicated by p53 phosphorylation status and apoptosis measured by DNA break frequency, was observed only in circadian-synchronized WT-MEF, not in CRYDKO or mouse hepatocarcinoma Hepa1c1c7 cells. These findings highlight that the DNA damage responses triggered by cisplatin are indeed governed by circadian control exclusively in clock-proficient cells. This outcome bears potential implications for enhancing or devising chronotherapy approaches for cancer patients.

Development of a rapid electrophoretic assay for genomic DNA damage quantification

Accuracy, sensitivity, simplicity, reproducibility, and low-cost are desirable requirements for genotoxicity assessment techniques. Here we describe a simple electrophoretic assay for genomic DNA lesions quantification (EAsy-GeL) based on subjecting DNA samples to rapid unwinding/renaturation treatments and neutral agarose gel electrophoresis. The experiments performed in this work involved different biological samples exposed to increasing environmental-simulated doses of ultraviolet-B (UVB) radiation, such as Escherichia coli, human leukocytes, and isolated human genomic DNA. DNA extraction was carried out using a universal and low-cost protocol, which takes about 4 h. Before electrophoresis migration, DNA samples were kept into a neutral buffer to detect double-strand breaks (DSBs) or subjected to a 5-min step of alkaline unwinding and neutral renaturation to detect single-strand breaks (SSBs) or incubated with the DNA repair enzyme T4-endonuclease V for the detection of cyclobutane pyrimidine dimers (CPDs) before the 5-min step of DNA unwinding/renaturation. Then, all DNA samples were separated by neutral agarose gel electrophoresis, the DNA average length of each lane was calculated through the use of free software, and the frequency of DNA breaks per kbp was determined by a simple rule of three. Dose-response experiments allowed the quantification of different levels of DNA damage per electrophoretic run, varying from a constant and low amount of DSBs/SSBs to high and dose-dependent levels of CPDs. Compared with other assays based on alkaline unwinding and gel electrophoresis, EAsy-GeL has important advantages for both environmental monitoring and laboratory testing purposes. The simplicity and applicability of this protocol to other types of DNA lesions, biological models, and agents make it ideal for genotoxicity, DNA repair studies, as well as for assessing exposure risks to ecosystems and human health.

Pluripotent stem cells with low differentiation potential contain incompletely reprogrammed DNA replication.

Reprogrammed pluripotent stem cells (PSCs) are valuable for research and potentially for cell replacement therapy. However, only a fraction of reprogrammed PSCs are developmentally competent. Genomic stability and accurate DNA synthesis are fundamental for cell development and critical for safety. We analyzed whether defects in DNA replication contribute to genomic instability and the diverse differentiation potentials of reprogrammed PSCs. Using a unique single-molecule approach, we visualized DNA replication in isogenic PSCs generated by different reprogramming approaches, either somatic cell nuclear transfer (NT-hESCs) or with defined factors (iPSCs). In PSCs with lower differentiation potential, DNA replication was incompletely reprogrammed, and genomic instability increased during replicative stress. Reprogramming of DNA replication did not correlate with DNA methylation. Instead, fewer replication origins and a higher frequency of DNA breaks in PSCs with incompletely reprogrammed DNA replication were found. Given the impact of error-free DNA synthesis on the genomic integrity and differentiation proficiency of PSCs, analyzing DNA replication may be a useful quality control tool.

GENOTOXIC AND PROTECTIVE ACTIVITY OF EXTRACTS FROM INULA BRITANNICA (FAM. COMPOSITAE)) IN THE BODY OF LABORATORY MICE

The protective and mutagen-modifying activity of the complex of biologically active substances(BAS) in the extract from the underground part of the plant Inula britannica in the cells of the visceral organs of laboratory mice was studied. Alkaline variation of the comet assay was used to determine genotoxic/antigenotoxic activity. The frequency of single-stranded DNA breaks in the cells of the brain andbone marrow, lungs, heart, kidneys, liver, stomach, spleen was assessed by the following parameters: %of DNA in tail, Olive tail moment, damage index. It was found that extracts of BAS in concentrations of100.0 mg/l and 150.0 mg/l did not show genotoxic activity, the frequency of single-strand breaks in allstudied organs did not statistically significantly exceed the spontaneous level in intact animals. Whenexposed to asymmetric dimethylhydrazine (UDMH), the cells of the studied organs showed a statisticallysignificant increase in the number of single-stranded DNA breaks compared to intact laboratory mice.When combined with UDMH as a positive control, extract of Inula britannica significantly reduces thegenotoxic effect of xenobiotics. The frequency of DNA breaks in animals that simultaneously receivedUDMH and Inula britannica extract was statistically significantly lower compared with mice that received only UDMH. All this suggests the presence of antimutagenic and antigenotoxic activity in theextract of Inula britannica.Key words: Inula britannica, biologically active substances, mutagen, antimutagen, genotoxicity,DNA comet assay.

Changes of Reactive Oxygen and Nitrogen Species and Mitochondrial Functioning in Human K562 and HL60 Cells Exposed to Ionizing Radiation

Free radicals generated by mitochondria are candidates for mediating long-lasting effects of radiation on cells, including genetic instability. To better understand the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in these long-term effects we assayed ROS and RNS levels, the mitochondrial membrane potential and mass, and the frequency of DNA strand breaks, apoptosis and necrosis in human leukemic cells (K562 and HL60) after 12 Gy of X irradiation. An increase in intracellular ROS level was observed immediately post-irradiation, and about 24 h later a second increase of ROS was accompanied by increase in nitrogen oxide, mitochondrial potential and mitochondrial mass in both cell types. The second peak of ROS level was partially inhibited by rotenone, an inhibitor of mitochondrial complex I, in K562 but not in HL60 cells suggesting that the sources of ROS differed in the two cell types. The frequency of DNA breaks showed kinetics similar to ROS levels, with a sharp peak immediately after irradiation and a second increase 24 and 48 h later, which was significantly higher in K562 cells. Forty-eight hours after irradiation an increase in the frequency of apoptotic cells was observed in both cell lines, which became larger and statistically significant in K562 cells after inhibition of mitochondrial complex I. Our results show that ionizing radiation activates cellular processes which produce long-lasting ROS and RNS radicals, which may have different sources in different cell types and could participate in cellular signaling networks important for radiosensitivity and mode of cell death.

The essential comet assay: a comprehensive guide to measuring DNA damage and repair

The comet assay (single cell gel electrophoresis) is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. The assay depends on the relaxation of supercoiled DNA in agarose-embedded nucleoids (the residual bodies remaining after lysis of cells with detergent and high salt), which allows the DNA to be drawn out towards the anode under electrophoresis, forming comet-like images as seen under fluorescence microscopy. The relative amount of DNA in the comet tail indicates DNA break frequency. The assay has been modified to detect various base alterations, by including digestion of nucleoids with a lesion-specific endonuclease. We describe here recent technical developments, theoretical aspects, limitations as well as advantages of the assay, and modifications to measure cellular antioxidant status and different types of DNA repair. We briefly describe the applications of this method in genotoxicity testing, human biomonitoring, and ecogenotoxicology.

The comet assay, DNA damage, DNA repair and cytotoxicity: hedgehogs are not always dead

DNA damage is commonly measured at the level of individual cells using the so-called comet assay (single-cell gel electrophoresis). As the frequency of DNA breaks increases, so does the fraction of the DNA extending towards the anode, forming the comet tail. Comets with almost all DNA in the tail are often referred to as 'hedgehog' comets and are widely assumed to represent apoptotic cells. We review the literature and present theoretical and empirical arguments against this interpretation. The level of DNA damage in these comets is far less than the massive fragmentation that occurs in apoptosis. 'Hedgehog' comets are formed after moderate exposure of cells to, for example, H2O2, but if the cells are incubated for a short period, 'hedgehogs' are no longer seen. We confirm that this is not because DNA has degraded further and been lost from the gel, but because the DNA is repaired. The comet assay may detect the earliest stages of apoptosis, but as it proceeds, comets disappear in a smear of unattached DNA. It is clear that 'hedgehogs' can correspond to one level on a continuum of genotoxic damage, are not diagnostic of apoptosis and should not be regarded as an indicator of cytotoxicity.

XPG and XPF Endonucleases Trigger Chromatin Looping and DNA Demethylation for Accurate Expression of Activated Genes

Nucleotide excision repair factors, initially characterized as part of DNA repair, have been shown to participate in the transcriptional process in the absence of genotoxic attack. However, their molecular function when recruited at the promoters of activated genes together with the transcription machinery remained obscure. Here we show that the NER factors XPG and XPF are essential for establishing CTCF-dependent chromatin looping between the promoter and terminator of the activated RARβ2 gene. Silencing XPG and/or XPF endonucleases, or mutations in their catalytic sites, prevents CTCF recruitment, chromatin loop formation, and optimal transcription of RARβ2. We demonstrated that XPG endonuclease promotes DNA breaks and DNA demethylation at promoters allowing the recruitment of CTCF and gene looping, which is further stabilized by XPF. Our results highlight a timely orchestrated activity of the NER factors XPG and XPF in the formation of the active chromatin hub that controls gene expression.

Radioprobing the conformation of DNA in a p53-DNA complex

Purpose: The frequency of DNA strand breaks produced by the decay of Auger electron-emitting radionuclides is inversely proportional to the distance of DNA nucleotides from the decay site; and thus is very sensitive to changes in the local conformation of the DNA. Analysis of the frequency of DNA breaks, or radioprobing, gives valuable information about the local DNA structure. More than 10 years ago, we demonstrated the feasibility of radioprobing using a DNA-repressor complex with a known structure. Herein, we used radioprobing to study the conformation of DNA in complex with the tumor suppressor protein 53 (p53). Several structures of p53-DNA complexes have been solved by X-ray crystallography. These structures, obtained with the p53 DNA binding domain, a truncated form, laid the groundwork for understanding p53-DNA interactions and their relation to p53 functions. However, whether all observed stereochemical details are relevant to the native p53-DNA complex remains unclear. A common theme of the crystallographic structures is the lack of significant bending in the central part of the DNA response element. In contrast, gel electrophoresis and electron microscopy data showed strong DNA bending and overtwisting upon binding to the native p53 tetramer.Methods: To analyze DNA in complex with p53, we incorporated 125I-dCTP in two different positions of synthetic duplexes containing the consensus p53-binding site.Results: The most significant changes in the break frequency distributions were detected close to the center of the binding site, which is consistent with an increase in DNA twisting in this region and local DNA bending and sliding.Conclusions: Our data confirm the main results of the studies made in solution and lay a foundation for systematic examination of interactions between DNA and native p53 using 125I radioprobing.

Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

Polζ ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability

Polζ is an error-prone DNA polymerase that is critical for embryonic development and maintenance of genome stability. To analyze its suggested role in somatic hypermutation (SHM) and possible contribution to DNA double-strand break (DSB) repair in class switch recombination (CSR), we ablated Rev3, the catalytic subunit of Polζ, selectively in mature B cells in vivo. The frequency of somatic mutation was reduced in the mutant cells but the pattern of SHM was unaffected. Rev3-deficient B cells also exhibited pronounced chromosomal instability and impaired proliferation capacity. Although the data thus argue against a direct role of Polζ in SHM, Polζ deficiency directly interfered with CSR in that activated Rev3-deficient B cells exhibited a reduced efficiency of CSR and an increased frequency of DNA breaks in the immunoglobulin H locus. Based on our results, we suggest a nonredundant role of Polζ in DNA DSB repair through nonhomologous end joining.

Single‐cell gel electrophoresis (the comet assay): Loops or fragments?

Single-cell gel electrophoresis, or the comet assay, is widely used to measure DNA damage and repair. Upon electrophoresis, the DNA of lysed, agarose-embedded cells known as nucleoids, extends towards the anode in a structure resembling a comet, the relative intensity of the tail reflecting the frequency of DNA breaks. The structural organization of the DNA within comet preparations is not fully understood. We have used fluorescent in situ hybridization with large-insert genomic probes and human Cot-I DNA to investigate whether the production of the comet tail is simply explained by the relaxation of supercoiled DNA loops. We find that, under neutral electrophoresis conditions, when the tail and head DNA are double-stranded, the probed sequence of DNA is seen as a linear array, consistent with extension from a fixed point on the nuclear core or matrix. After alkaline electrophoresis, the appearance of the fluorescent probes suggests that linear DNA has coalesced into a granular form.

Altered gene expression profiles and higher frequency of spontaneous DNA strand breaks in APEX2-null thymus

A second class II AP endonuclease, APEX2, possesses strong 3′–5′ exonuclease and 3′-phosphodiesterase activities but only very weak AP-endonuclease activity. APEX2 associates with proliferating cell nuclear antigen (PCNA), and the progression of S phase of the cell cycle is accompanied by its expression. APEX2-null mice exhibit severe dyslymphopoiesis in thymus as well as moderate dyshematopoiesis and growth retardation. Comparative gene expression profiling of wild-type and APEX2-null mice using an oligonucleotide microarray revealed that APEX2-null thymus has significantly altered gene expression profiles, reflecting its altered populations of thymocytes. Beyond these altered populations, APEX2-null thymus exhibits significant alterations in expression of genes involved in DNA replication, recombination and repair, including Apex1, Exo1 and Fen1 as well as master genes for the DNA damage response, such as E2f1, Chek1, and proapoptotic genes. We therefore examined the extent of DNA strand breakage, and found that both of single-strand breaks detected as comets and double-strand breaks detected as γH2AX foci were significantly higher in frequency in most APEX2-null thymocytes compared to wild-type thymocytes. This higher frequency of DNA breaks was accompanied by increased expression of PCNA and increased phosphorylation of p53 at Ser23 and to a lesser extent, at Ser18. The present study clearly demonstrates that APEX2-null lymphocytes have a higher frequency of DNA breaks, indicating that APEX2 may play an important role(s) during their generation and/or repair.

Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity

Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5–10 mM), hydrazine (HZ; 0.5–4 mM), cadmium sulfate (CD; 31.2 and 62.5 μM), 4,4′-methylene bis (2-chloroaniline) (MOCA; 31.2–125 μM), isobutyl nitrite (IBN; 7.8–31.2 μM) and tetranitromethane (TNM; 1.9–15.6 μM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats.

Mouse strains with an active H2-Ea meiotic recombination hot spot exhibit increased levels of H2-Ea-specific DNA breaks in testicular germ cells.

We devised a sensitive method for the site-specific detection of rare meiotic DNA strand breaks in germ cell-enriched testicular cell populations from mice that possess or lack an active recombination hot spot at the H2-Ea gene. Using germ cells from adult animals, we found an excellent correlation between the frequency of DNA breaks in the 418-bp H2-Ea hot spot and crossover activity. The temporal appearance of DNA breaks was also studied in 7- to 18-day-old mice with an active hot spot during the first waves of spermatogenesis. The number of DNA breaks detected rose as leptotene and zygotene spermatocytes populate the testis with a peak at day 14 postpartum, when leptotene, zygotene, and early pachytene spermatocytes are the most common meiotic prophase I cell types. The number of DNA breaks drops precipitously 1 day later, when middle to late pachytene spermatocytes become the dominant subtype. The recombination-related breaks in the hot spot likely reflect SPO11-induced double-strand breaks and/or recombination intermediates containing free 3' hydroxyl groups.

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 1 4 and 1 2 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 1 2 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.

Ethanol-induced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: effects of acute and chronic ethanol exposure.

Ethanol was given to male Wistar rats as an acute dose (5 g/kg) or continuously at 5% (w/v) in a liquid diet to provide 36% of the caloric requirement. Free radicals generated in the liver were collected as a stable adduct in bile following the in vivo administration of the spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN; 700 mg/kg). [1-(13)C]ethanol was used to confirm the formation of the 1-hydroxyethyl radical and to demonstrate that this was ethanol-derived in the case of the single-dose treatment. Free radical production increased up to 1h after the acute dose and then plateaued over the next 30 min. During chronic exposure to ethanol, free radical generation increased significantly after 1 week and then declined again to remain at a low level over the next 2 weeks; this transient increase corresponded closely with the induction of cytochrome P-450 2E1 (CYP 2E1) in response to ethanol feeding. Single-cell electrophoresis was used to investigate effects on DNA. After an acute dose of ethanol, the frequency of single-strand breaks increased from 1 h to peak at 6 h but then declined again to control values by 12 h. During the chronic exposure, an increase in the frequency of DNA breaks was seen at 3 days, reached a peak at 1 week and then decreased slowly over the next 5 weeks. The effects of antioxidants on these parameters was investigated after an acute dose of ethanol. Pre-treatment with vitamin C (400 mg/kg, i.p., daily for 5 days) or vitamin E (100 mg/kg, i.p., for 5 days) prior to the administration of ethanol (5 g/kg) inhibited generation of the 1-hydroxyethyl-POBN adduct by 30 and 50%, respectively, and both agents prevented the increased frequency of DNA single-strand breaks caused by ethanol. The significance of the temporal coincidence of changes in the above parameters in response to ethanol is discussed.

Activation of bullous pemphigoid antigen gene in mouse ear epidermis by ultraviolet radiation.

Bullous pemphigoid (BP) is an autoimmune blistering disease and is a photoaggravated dermatosis, but the mechanism of the aggravation is still unknown. Since damage to DNA initiates transcription of some genes, we investigated in epidermis of mouse ears the relationship between DNA damage by ultraviolet (UV) radiation and BP antigen (BP-Ag) gene activation. For this, albino male mice were irradiated with 254 nm wavelength UV for a total dose of 500 J m-2. At fixed times (0.5, 2, 24, 48 and 72 h) post-UV irradiation, mouse ears were cut off, frozen and sectioned. In the sections, it was found that immunohistochemically detectable pyrimidine dimers were observed in nuclei of all epidermal cells at 0.5 h that were almost repaired by 72 h; a frequency of single strand breaks in DNA detected by in situ nick translation started to increase in nuclei of all epidermal cell layers at 0.5 h and the increase continued up to 24 h; mRNA for BP-Ag localized by non-radioactive in situ hybridization appeared in nuclei of basal cells at 0.5 h and in both nuclei and cytoplasm at 2 h; and immunoreactive BP-Ag started to increase in the basal cell cytoplasm and in the basement membrane zone at 2 h. BP-Ag started to accumulate in the basement membrane zone at 2 h. It is suggested that UV radiation increased BP-Ag synthesis through BP-Ag synthesis through BP-Ag gene activation and that this reaction is a factor which aggravates BP following UV irradiation in BP patients.

Studies on α-Hexachlorocyclohexane Cytotoxicity, Genotoxicity and Cytochrome P450 Induction in Primary Hepatocytes and Hepatoma Cell Lines from Rodents and Humans

α-Hexachlorocyclohexane (α-HCH) was examined for cytotoxicity, genotoxicity and cytochrome P450 induction in primary cultures of mouse, rat and human hepatocytes and in three hepatoma cell lines (Hepa 1c1c7, FaO and Hep G2, from mouse, rat and man, respectively). The cell lines were much more sensitive to the cytotoxicity of the classical inducers phenobarbital and 3-methylcholanthrene than that of α-HCH, whereas no cytotoxicity was observed in primary hepatocytes. Exposure for 24 hours to 0.32mM α-HCH produced a modest, but statistically significant, frequency of DNA breaks, as measured by the alkaline elution assay, in the mouse Hepa 1c1c7 cell line, and the human Hep G2 cell line, but not in the rat FaO cell line. In the Hep G2 cell line, the amount of DNA fragmentation was found to increase with the length of exposure. Compared with the results of previous observations on primary cultures, with regard to species specificity, only the human cell line gave a concordant positive response. Monooxygenase activity induction in primary hepatocytes, despite rather high initial levels of 7-ethoxycoumarin-O-deethylase activity, was low with the classical inducers phenobarbital and 3-methylcholanthrene. α-HCH caused no induction of monooxygenase. The rat FaO and human Hep G2 cell lines were sensitive to α-HCH, but only after long exposure. The results of this study support the hypothesis that α-HCH might act as a weak genotoxic agent in humans, but they also suggest caution in the extrapolation to the in vivo situation of the observations made in established cell lines.

Testing of p-dichlorobenzene and hexachlorobenzene for their ability to induce DNA damage and micronucleus formation in primary cultures of rat and human hepatocytes.

The genotoxicity of p-dichlorobenzene (DCB) and hexachlorobenzene (HCB) was evaluated in primary cultures of rat and human hepatocytes. DNA fragmentation was measured by the alkaline elution technique and clastogenic activity by the increase in micronucleus formation. In rat hepatocytes, exposure to concentrations of DCB ranging from 0.56 to 3.2 mM and of HCB from 0.1 to 0.56 mM did not induce any significant increase in the frequency of DNA breaks but consistently produced a statistically significant increase in the frequency of micronucleated cells. In human hepatocytes, under the same experimental conditions, the response to DCB was negative in terms of both DNA fragmentation and clastogenic effect, whereas HCB produced a significant increase in the frequency of both DNA breaks and micronuclei. Taken as a whole these results suggest that of the two pesticides examined only HCB should be considered as a weak genotoxic carcinogen.