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Background Cisplatin is a primary chemotherapy choice for various solid tumors. DNA damage caused by cisplatin results in apoptosis of tumor cells. Cisplatin‐induced DNA damage, however, may also result in mutations in normal cells and the initiation of secondary malignancies. In the current study, we have used the erythrocyte PIG‐A assay to evaluate mutagenesis in non‐tumor hematopoietic tissue of cancer patients receiving cisplatin chemotherapy. Methods Twenty‐one head and neck cancer patients undergoing treatment with cisplatin were monitored for the presence of PIG‐A mutant total erythrocytes and the young erythrocytes, reticulocytes (RETs), in peripheral blood for up to five and a half months from the initiation of the anti‐neoplastic chemotherapy. Results PIG‐A mutant frequency (MF) in RETs increased at least two‐fold in 15 patients at some point of the monitoring, while the frequency of total mutant RBCs increased at least two‐fold in 6 patients. A general trend for an increase in the frequency of mutant RETs and total mutant RBCs was observed in 19 and 18 patients, respectively. Only in one patient did both RET and total RBC PIG‐A MFs did not increase at any time‐point over the monitoring period. Conclusion Cisplatin chemotherapy induces moderate increases in the frequency of  PIG‐A mutant erythrocytes in head and neck cancer patients. Mutagenicity measured with the flow cytometric PIG‐A assay may serve as a tool for predicting adverse outcomes of genotoxic antineoplastic therapy. Cisplatin chemotherapy induces moderate increases in the frequency of PIG‐A mutant erythrocytes in head and neck cancer patients. Mutagenicity measured with the flow cytometric PIG‐A assay may serve as a tool for predicting adverse outcomes of genotoxic antineoplastic therapy.

In contrast to somatic mutations, mutations in germ cells affect every cell of any organism derived from the germ cell and therefore are related to numerous genetic diseases. However, there is no suitable assay to evaluate the mutagenic sensitivities of both male and female germ cells. The main type of Caenorhabditis elegans ( C. elegans ) is hermaphroditic, where spermatogenesis and oogenesis occur chronologically at specific stages, allowing induction of mutations in either sperm or eggs exclusively. In this study, we used the alkylating agent ethyl methanesulfonate and N -ethyl- N -nitrosourea to induce germline mutations in C. elegans at different developmental stages and analyzed mutation frequency and mutational spectrum from data gathered using next-generation sequencing (NGS) technology. Our results revealed low spontaneous mutation rates of C. elegans , along with distinct mutagenic effects elicited by the two mutagens. Our data show that the parental worms treated during germ cell mitosis, spermatogenesis, and oogenesis resulted in different mutation frequencies in their offspring, and female germ cells could be very susceptible to mutagen exposure during oogenesis. In summary, our study indicates that the use of C. elegans and its specific chronological hermaphroditism would be a promising way to explore the sensitivities of both male and female germ cells to mutagens.

In vitro genotoxicity testing that employs metabolically active human cells may be better suited for evaluating human in vivo genotoxicity than current bacterial or non-metabolically active mammalian cell systems. In the current study, 28 compounds, known to have different genotoxicity and carcinogenicity modes of action (MoAs), were evaluated over a wide range of concentrations for the ability to induce DNA damage in human HepG2 and HepaRG cells. DNA damage dose–responses in both cell lines were quantified using a combination of high-throughput high-content (HTHC) CometChip technology and benchmark dose (BMD) quantitative approaches. Assays of metabolic activity indicated that differentiated HepaRG cells had much higher levels of cytochromes P450 activity than did HepG2 cells. DNA damage was observed for four and two out of five indirect-acting genotoxic carcinogens in HepaRG and HepG2 cells, respectively. Four out of seven direct-acting carcinogens were positive in both cell lines, with two of the three negatives being genotoxic mainly through aneugenicity. The four chemicals positive in both cell lines generated HTHC Comet data in HepaRG and HepG2 cells with comparable BMD values. All the non-genotoxic compounds, including six non-genotoxic carcinogens, were negative in HepaRG cells; five genotoxic non-carcinogens also were negative. Our results indicate that the HTHC CometChip assay detects a greater proportion of genotoxic carcinogens requiring metabolic activation (i.e., indirect carcinogens) when conducted with HepaRG cells than with HepG2 cells. In addition, BMD genotoxicity potency estimate is useful for quantitatively evaluating CometChip assay data in a scientifically rigorous manner.

Primary human hepatocytes (PHHs) are considered the “gold standard” for evaluating hepatic metabolism and toxicity of xenobiotics. In the present study, we evaluated the genotoxic potential of four indirect-acting (requiring metabolic activation) and six direct-acting genotoxic carcinogens, one aneugen, and five non-carcinogens that are negative or equivocal for genotoxicity in vivo in cryopreserved PHHs derived from three individual donors. DNA damage was determined over a wide range of concentrations using the CometChip technology and the resulting dose–responses were quantified using benchmark dose (BMD) modeling. Following a 24-h treatment, nine out of ten genotoxic carcinogens produced positive responses in PHHs, while negative responses were found for hydroquinone, aneugen colchicine and five non-carcinogens. Overall, PHHs demonstrated a higher sensitivity (90%) for detecting DNA damage from genotoxic carcinogens than the sensitivities previously reported for HepG2 (60%) and HepaRG (70%) cells. Quantitative analysis revealed that most of the compounds produced comparable BMD10 values among the three types of hepatocytes, while PHHs and HepaRG cells produced similar BMD1SD values. Evidence of sex- and ethnicity-related interindividual variation in DNA damage responses was also observed in the PHHs. A literature search for in vivo Comet assay data conducted in rodent liver tissues demonstrated consistent positive/negative calls for the compounds tested between in vitro PHHs and in vivo animal models. These results demonstrate that CometChip technology can be applied using PHHs for human risk assessment and that PHHs had higher sensitivity than HepaRG cells for detecting genotoxic carcinogens in the CometChip assay.

Biomarker strategies are increasingly being applied in drug development to address the challenges posed by heterogeneity in the underlying mechanisms of disease processes and inter-patient variability in drug responses. With the aim of enhancing understanding of the regulatory significance of such biomarker data by regulators and sponsors, the US FDA initiated a programme in 2004 to allow sponsors to submit exploratory data voluntarily, without immediate regulatory impact. This article discusses a selection of case studies from the first 5 years of this programme, highlighting lessons learned. Heterogeneity in the underlying mechanisms of disease processes and inter-patient variability in drug responses are major challenges in drug development. To address these challenges, biomarker strategies based on a range of platforms, such as microarray gene-expression technologies, are increasingly being applied to elucidate these sources of variability and thereby potentially increase drug development success rates. With the aim of enhancing understanding of the regulatory significance of such biomarker data by regulators and sponsors, the US Food and Drug Administration initiated a programme in 2004 to allow sponsors to submit exploratory genomic data voluntarily, without immediate regulatory impact. In this article, a selection of case studies from the first 5 years of this programme — which is now known as the voluntary exploratory data submission programme, and also involves collaboration with the European Medicines Agency — are discussed, and general lessons are highlighted.

Abstract Tert-butylhydroquinone (TBHQ) is a monofunctional Phase II enzyme inducer, which produces reactive oxygen species. Incubation with a sublethal concentration of TBHQ increased the activities of both γ-glutamyl transpeptidase (GGT) and γ-glutamylcysteine synthetase (GCS), although the mechanisms are different (Liu and colleagues, accompanying manuscript). In this study, we found that TBHQ increased intracellular glutathione (GSH) content in rat lung epithelial L2 cells. L2 cells pretreated with a nontoxic concentration of TBHQ (50 microM) acquired resistance to a subsequent challenge with a normally lethal concentration of TBHQ (200 microM). Pretreatment with L-buthionine S,R-sulfoximine (BSO), an inhibitor of GCS, prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Similarly, pretreatment with acivicin, an inhibitor of GGT, also prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Nevertheless, blockage of GGT by acivicin could be bypassed using 2-oxothiazolidine-4-carboxylate (procysteine) to provide the cell with a source of cysteine. This allowed an increase in GSH and restored resistance in the TBHQ-pretreated cells. The results suggest that elevation of GCS and GGT activities participated in acquired resistance to quinone toxicity.

Abstract Sublethal quinone-mediated oxidative stress stimulates increases in the activities and mRNA levels of γ-glutamyl transpeptidase (GGT) and γ-glutamylcysteine synthetase (GCS) in rat lung epithelial L2 cells [Kugelman, A. et al. 1994. Am. J. Respir. Cell Mol. Biol. 11:586-592; Shi, M. M. et al. 1994. J. Biol. Chem. 269:26512-26517]. The present study demonstrated that the quinone-induced increases in these two enzymes were differentially regulated. L2 cells were exposed to various concentrations of tertiary-butylhydroquinone (TBHQ) for different periods of times. TBHQ increased the activities and the mRNAs for GGT and the catalytic subunit of GCS; however, the time- and concentration-dependencies differed. With 50 microM TBHQ, GCS activity increased significantly by 6 h whereas the activity of GGT was not increased until later. Under the same conditions, the highest GCS-mRNA level observed was at 6 h whereas the mRNA level of GGT increased after 6 h, reached a higher level at 12 h, and then returned to the control level by 24 h. Differences were also observed in the concentration-dependence of mRNA increases between the GGT and GCS. Actinomycin D (an inhibitor of RNA synthesis) abolished the increase of GCS-mRNA but not the increase in GGT-mRNA, suggesting a difference in regulation by TBHQ between these two genes. Nuclear run-on experiments confirmed that the increase of GCS-mRNA, but not GGT-mRNA was due to increased transcription. The increase in GGT-mRNA probably results from a decreased degradation rate. The differences between these two enzymes demonstrate how cells can use multiple mechanisms for regulating gene expression in response to oxidative stress.

Pulmonary injury from nitrogen dioxide ( NO2) may in part be related to the generation of aldehydic compounds, which bind with cellular proteins and subsequently impair or inhibit cell function. We examined the generation of aldehydes from guinea pig tracheobronchial epithelial (GPTE) cell monolayers exposed to NO2. with the use of dinitrophenylhydrazine (DNP) to derivatize aldehydic compounds, glycolaldehyde, a two carbon α-hydroxyaldehyde, was identified in elevated levels in the basolateral fluid from monolayers exposed to NO2. DNP-glycolaldehyde levels were 81.2 ± 2.7 and 234.0 ± 42.6 nM in response to a 1-hr exposure to 1 and 5 ppm NO2, respectively, as compared to an air-control value of 20.3 ± 6.8 nM. Taking into account dilution and reactivity, cellular glycolaldehyde levels could have reached as high as 3 mM for the 60-min exposure period (i.e., 0.05 mM/min). The effects of exogenous glycolaldehyde on GPTE ouabain-sensitive basolateral 86 Rb uptake (an index of Na+, K+- ATPase activity) were examined and compared with the actions of NO2 exposure. Bolus addition of glycolaldehyde to the basolateral fluid at concentrations ≥5 mM led to an inhibition of ouabain-sensitive 86 Rb uptake, while lower concentrations had no effect. The effects of exogenous glycolaldehyde differ from NO2 exposure, which led to a sustained elevation of ouabain-sensitive 86 Rb uptake with presumed generation of glycolaldehyde at a continuous low level. Glycolaldehyde does not appear to play a significant role in the acute alterations of sodium pump activity, suggesting that the NO2-induced changes in Na+, K+- ATPase activity of GPTE monolayers probably are further mediated by other lipid peroxidation products/oxidation processes yet to be identified.

Chronic lung diseases of the newborn may result from inflammation or oxygen therapy, a procedure aimed at maintaining adequate oxygen delivery to the brain and other organs. Both of these causes of chronic lung disease would be expected to involve injury from reactive oxygen metabolites. The literature abounds with reviews on the production of reactive oxygen species by inflammatory cells and pulmonary oxygen toxicity as a consequence of oxygen therapy. Inflammatory cells produce reactive oxygen species as part of their microbicidal action. This can unfortunately injure normal tissue as collateral damage in the war against bacteria. Oxygen toxicity likely originates from increased generation of hydrogen peroxide by the mitochondria and perhaps other organelles at high oxygen concentrations. In this chapter, we have described methods for evaluating the involvement of reactive oxygen species. As the reader will find inherent difficulties in measurement of reactive oxygen species makes assessment of the contribution of these species to diseases, such as bronchopulmonary dysplasia, a monumental task. Fortunately, some of the newer methods described below may lead the way toward a more rigorous evaluation of cause and effect between chronic diseases of early infancy and production of reactive oxygen species.

Pulmonary injury from nitrogen dioxide (NO2) may in part be related to the generation of aldehydic compounds, which bind with cellular proteins and subsequently impair or inhibit cell function. We examined the generation of aldehydes from guinea pig tracheobronchial epithelial (GPTE) cell monolayers exposed to NO2. With the use of dinitrophenylhydrazine (DNP) to derivatize aldehydic compounds, glycolaldehyde, a two carbon alpha-hydroxyaldehyde, was identified in elevated levels in the basolateral fluid from monolayers exposed to NO2. DNP-glycolaldehyde levels were 81.2 +/- 2.7 and 234.0 +/- 42.6 nM in response to a 1-hr exposure to 1 and 5 ppm NO2, respectively, as compared to an air-control value of 20.3 +/- 6.8 nM. Taking into account dilution and reactivity, cellular glycolaldehyde levels could have reached as high as 3 mM for the 60-min exposure period (i.e., 0.05 mM/min). The effects of exogenous glycolaldehyde on GPTE ouabain-sensitive basolateral 86Rb uptake (an index of Na+,K(+)-ATPase activity) were examined and compared with the actions of NO2 exposure. Bolus addition of glycolaldehyde to the basolateral fluid at concentrations > or = 5 mM led to an inhibition of ouabain-sensitive 86Rb uptake, while lower concentrations had no effect. the effects of exogenous glycolaldehyde differ from NO2 exposure, which led to a sustained elevation of ouabain-sensitive 86Rb uptake with presumed generation of glycolaldehyde at a continuous low level. Glycolaldehyde does not appear to play a significant role in the acute alterations of sodium pump activity, suggesting that the NO2-induced changes in Na+,K(+)-ATPase activity of GPTE monolayers probably are further mediated by other lipid peroxidation products/oxidation processes yet to be identified.