Paleogenomics illuminates the evolutionary history of the extinct Holocene "horned" crocodile of Madagascar, Voay robustus.

Ancient DNA is transforming our ability to reconstruct historical patterns and mechanisms shaping modern diversity and distributions. In particular, molecular data from extinct Holocene island faunas have revealed surprising biogeographic scenarios. Here, we recovered partial mitochondrial (mt) genomes for 1300-1400 year old specimens (n = 2) of the extinct "horned" crocodile, Voay robustus, collected from Holocene deposits in southwestern Madagascar. Phylogenetic analyses of partial mt genomes and tip-dated timetrees based on molecular, fossil, and stratigraphic data favor a sister group relationship between Voay and Crocodylus (true crocodiles). These well supported trees conflict with recent morphological systematic work that has consistently placed Voay within Osteolaeminae (dwarf crocodiles and kin) and provide evidence for likely homoplasy in crocodylian cranial anatomy and snout shape. The close relationship between Voay and Crocodylus lends additional context for understanding the biogeographic origins of these genera and refines competing hypotheses for the recent extinction of Voay from Madagascar.

Standardized cell sources and recommendations for good cell culture practices in genotoxicity testing.

Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.

Vehicle and positive control values from the in vivo rodent comet assay and biomonitoring studies using human lymphocytes: historical database and influence of technical aspects.

There is increased interest in the in vivo comet assay in rodents as a follow-up approach for determining the biological relevance of chemicals that are genotoxic in in vitro assays. This is partly because, unlike other assays, DNA damage can be assessed in this assay in virtually any tissue. Since background levels of DNA damage can vary with the species, tissue, and cell processing method, a robust historical control database covering multiple tissues is essential. We describe extensive vehicle and positive control data for multiple tissues from rats and mice. In addition, we report historical data from control and genotoxin-treated human blood. Technical issues impacting comet results are described, including the method of cell preparation and freezing. Cell preparation by scraping (stomach and other GI tract organs) resulted in higher % tail DNA than mincing (liver, spleen, kidney etc) or direct collection (blood or bone marrow). Treatment with the positive control genotoxicant, ethyl methanesulfonate (EMS) in rats and methyl methanesulfonate in mice, resulted in statistically significant increases in % tail DNA. Background DNA damage was not markedly increased when cell suspensions were stored frozen prior to preparing slides, and the outcome of the assay was unchanged (EMS was always positive). In conclusion, historical data from our laboratory for the in vivo comet assay for multiple tissues from rats and mice, as well as human blood show very good reproducibility. These data and recommendations provided are aimed at contributing to the design and proper interpretation of results from comet assays.

Xenobiotic metabolism gene expression in the EpiDermin vitro 3D human epidermis model compared to human skin.

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm model appears to be representative of human skin. Models such as EpiDerm provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.

Hybridization leads to host-use divergence in a polyphagous butterfly sibling species pair.

Climate warming has lead to increased genetic introgression across a narrow hybrid zone separating the eastern and Canadian tiger swallowtails (Papilio glaucus and Papilio canadensis). This situation has led to the formation of an allochronically separated hybrid population with a delayed emerging phenotype or "late flight". Here, we assess how the recombination of the parental genomes that lead to this phenotype may have facilitated another major ecological shift, host-use divergence. We first contrast the ovipositional profiles of the late flight population to that of the parental species P. glaucus and P. canadensis. Subsequently we contrast the larval survival and growth of the late flight, a P. canadensis and a P. glaucus population, and a population from the northern edge of the hybrid zone on five hosts. Our results indicate that the ovipositional preference of this hybrid swarm is identical to that of the introgressing parental species, P. glaucus. Due to the absence of the preferred hosts of P. glaucus (Liriodendron tulipifera L. and Ptelea trifoliata L.) where the late flight occurs, this ovipositional pattern implies a functional specialization onto a secondary host of both parental species, Fraxinus americana L. In contrast, the larval host-use abilities represent a mixture of P. glaucus and P. canadensis, indicating divergence in larval host-use abilities has not taken place. However, high genetic variability (genetic coefficient of variation) is present for growth on F. americana in the late flight hybrid swarm and tradeoffs for larval performance on the preferred hosts of the parental species are evident; indicating a strong potential for future specialization in larval host-use abilities. This current scenario represents an instance where a shift in a major ecological trait, host use, is likely occurring as a byproduct of a shift in an unrelated trait (delayed emergence) leading to partial reproductive isolation.

In vitro approaches to develop weight of evidence (WoE) and mode of action (MoA) discussions with positive in vitro genotoxicity results.

A recent analysis by Kirkland et al. [Kirkland, D., Aardema, M., Henderson, L. and Müller, L. (2005) Evaluation of the ability of a battery of 3 in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat. Res. 584, 1-256] demonstrated an extremely high false positive rate for in vitro genotoxicity tests when compared with carcinogenicity in rodents. In many industries, decisions have to be made on the safety of new substances, and health risk to humans, without rodent carcinogenicity data being available. In such cases, the usual way to determine whether a positive in vitro genotoxicity result is relevant (i.e. indicates a hazard) for humans is to develop weight of evidence (WoE) or mode of action (MoA) arguments. These are based partly on further in vitro investigations, but usually rely heavily on tests for genotoxicity in one or more in vivo assays. However, for certain product types in the European Union, the use of animals for genotoxicity testing (as well as for other endpoints) will be prohibited within the next few years. Many different examples have been described that indicate DNA damage and genotoxic responses in vitro can arise through non-relevant in vitro events that are a result of the test systems and conditions used. The majority of these non-relevant in vitro events can be grouped under a category of 'overload of normal physiology' that would not be expected to occur in exposed humans. However, obtaining evidence in support of such MoAs is not easy, particularly for those industries prohibited from carrying out in vivo testing. It will become necessary to focus on in vitro studies to provide evidence of non-DNA, threshold or in vitro-specific processes and to discuss the potential for such genotoxic effects to occur in exposed humans. Toward this end, we surveyed the published literature for in vitro approaches that may be followed to determine whether a genotoxic effect observed in vitro will occur in humans. Unfortunately, many of the approaches we found are based on only a few published examples and validated approaches with consensus recommendations often do not exist. This analysis highlights the urgent need for developing consensus approaches that do not rely on animal studies for dealing with in vitro genotoxins.

Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing.

This report summarizes the proceedings of the September 9-10, 2005 meeting of the Expert Working Group on Hazard Identification and Risk Assessment in Relation to In Vitro Testing, part of an initiative on genetic toxicology. The objective of the Working Group was to develop recommendations for interpretation of results from tests commonly included in regulatory genetic toxicology test batteries, and to propose an appropriate strategy for follow-up testing when positive in vitro results were obtained in these assays. The Group noted the high frequency of positive in vitro findings in the genotoxicity test batteries with agents found not to be carcinogenic and thought not to pose a carcinogenic health hazard to humans. The Group agreed that a set of consensus principles for appropriate interpretation and follow-up testing when initial in vitro tests are positive was needed. Current differences in emphasis and policy among different regulatory agencies were recognized as a basis of this need. Using a consensus process among a balanced group of recognized international authorities from industry, government, and academia, it was agreed that a strategy based on these principles should include guidance on: (1) interpretation of initial results in the "core" test battery; (2) criteria for determining when follow-up testing is needed; (3) criteria for selecting appropriate follow-up tests; (4) definition of when the evidence is sufficient to define the mode of action and the relevance to human exposure; and (5) definition of approaches to evaluate the degree of health risk under conditions of exposure of the species of concern (generally the human). A framework for addressing these issues was discussed, and a general "decision tree" was developed that included criteria for assessing the need for further testing, selecting appropriate follow-up tests, and determining a sufficient weight of evidence to attribute a level of risk and stop testing. The discussion included case studies based on actual test results that illustrated common situations encountered, and consensus opinions were developed based on group analysis of these cases. The Working Group defined circumstances in which the pattern and magnitude of positive results was such that there was very low or no concern (e.g., non-reproducible or marginal responses), and no further testing would be needed. This included a discussion of the importance of the use of historical control data. The criteria for determining when follow-up testing is needed included factors, such as evidence of reproducibility, level of cytotoxicity at which an increased DNA damage or mutation frequency is observed, relationship of results to the historical control range of values, and total weight of evidence across assays. When the initial battery is negative, further testing might be required based on information from the published literature, structure activity considerations, or the potential for significant human metabolites not generated in the test systems. Additional testing might also be needed retrospectively when increase in tumors or evidence of pre-neoplastic change is seen. When follow-up testing is needed, it should be based on knowledge about the mode of action, based on reports in the literature or learned from the nature of the responses observed in the initial tests. The initial findings, and available information about the biochemical and pharmacological nature of the agent, are generally sufficient to conclude that the responses observed are consistent with certain molecular mechanisms and inconsistent with others. Follow-up tests should be sensitive to the types of genetic damage known to be capable of inducing the response observed initially. It was recognized that genotoxic events might arise from processes other than direct reactivity with DNA, that these mechanisms may have a non-linear, or threshold, dose-response relationship, and that in such cases it may be possible to determine an exposure level below which there is negligible concern about an effect due to human exposures. When a test result is clearly positive, consideration of relevance to human health includes whether other assays for the same endpoint support the results observed, whether the mode or mechanism of action is relevant to the human, and - most importantly - whether the effect observed is likely to occur in vivo at concentrations expected as a result of human exposure. Although general principles were agreed upon, time did not permit the development of recommendations for the selection of specific tests beyond those commonly employed in initial test batteries.

Second trimester Doppler ultrasound screening of the uterine arteries differentiates between subsequent normal and poor outcomes of hypertensive pregnancy: two different pathophysiological entities?

The 'classical' concept that pregnancy-induced hypertension (PIH) and pre-eclampsia (PE) primarily originate from defective placentation in early pregnancy has been challenged recently. There is growing evidence that other factors, including maternal predisposing conditions, also play a significant role in the pathophysiology of PIH and PE. The aim of the present study was to test the hypothesis that PIH and PE with an early onset and poor pregnancy outcome is associated with defective placentation, e.g. inadequate spiral artery dilatation and subsequent reduced uteroplacental perfusion, whereas PIH and PE with normal pregnancy outcome is not. Using Doppler ultrasound, we measured the uterine artery pulsatility index (PI) in a population of 531 nulliparous women in the 22nd week of gestation. Uterine artery PI was used as an index of resistance to blood flow in the uteroplacental circulation. Outcome measures were PIH/PE with or without poor pregnancy outcome, preterm birth and intra-uterine growth restriction (IUGR). The results revealed a striking difference between PI values for PIH/PE with and without poor pregnancy outcome. Uterine artery PI in the 22nd week was increased significantly in pregnancies which developed early-onset (before 35 weeks) PIH/PE with a poor pregnancy outcome. In contrast, uterine artery PI values were normal in women who developed PIH/PE, but had a good pregnancy outcome. There was a significant correlation between 22nd week uterine artery PI and subsequent preterm birth or IUGR. Our results indicate that only PIH/PE with poor pregnancy outcome is associated with defective placentation, whereas PIH/PE with good outcome is not. These findings support the concept of heterogeneous causes of hypertensive disorders of pregnancy.

Photoclastogenicity-an improved protocol, its validation, and investigation of the photogenotoxicity of DMBA.

An improved protocol was developed to detect light-induced clastogenic photoproducts in Chinese hamster ovary (CHO) cells. Dishes (60 mm) containing cells and the test material or vehicle control in 3 mL of phosphate-buffered saline were exposed to light using a SUNTEST CPS solar simulation unit. Importantly, cells were exposed at about 25 cm from the light source, thereby allowing a short exposure time of 2 min. With this exposure the assay was conducted with lids removed during the UV exposure with minimal risk of contamination. After preliminary experiments an exposure of 165.6 mJ/cm(2) UVA: 17.0 mJ/cm(2) UVB was selected for treatments with the different phototoxins. Under these exposure conditions about 10-15% aberrant cells were induced in vehicle control cultures with no or minimal cytotoxicity. The well-known photoclastogens 8-methoxypsoralen (8-MOP) and chlorpromazine (CLZ) were tested. In agreement with published data, 8-MOP and CLZ were clastogenic (lowest observed effect level, LOEL, was 0.0159 microg/mL and 1.03 microg/mL, respectively). In the absence of UV, 8-MOP was clastogenic at a much higher concentration (LOEL 251 microg/mL without UV vs. 0.0159 microg/mL with UV) while CLZ was negative up to a toxic concentration of 35 microg/mL. 7,12-Dimethylbenz[a]anthracene (DMBA), which is photomutagenic in bacteria, was clastogenic at > or =0.005 microg/mL with UV light (without S9) and at > or =2.53 microg/mL with S9 (without UV light). These results demonstrate the utility of the protocol for the detection of photoclastogenicity and expand the characterization of DMBA's photogenotoxic activity.

Uterine artery Doppler flow and uteroplacental vascular pathology in normal pregnancies and pregnancies complicated by pre-eclampsia and small for gestational age fetuses.

This study was conducted to investigate the association between uterine artery Doppler flow patterns and uteroplacental vascular pathology in normal and complicated pregnancies in view of the recently described concept of heterogeneous causes of hypertensive pregnancy complications. Forty-three women whose pregnancies were complicated by pre-eclampsia, the HELLP (Haemolysis, Elevated Liver enzymes, Low Platelets) syndrome and/or small for gestational age (SGA) fetuses and 27 women with normal pregnancies undergoing elective caesarean section were included. We obtained uterine artery Doppler waveforms at a mean of 4 days before delivery. Placental bed biopsies were obtained at caesarean section and analysed for physiological changes and pathological changes. We found that abnormal uterine artery Doppler flow was strongly associated with pregnancy complications. Absence of physiological changes was seen in 58 per cent of complicated pregnancies and 40 per cent of normal pregnancies. Pathological changes were seen in 58 per cent of complicated pregnancies and 53 per cent of normal pregnancies; they occurred in spiral arteries with and without physiological changes, and there was no significant correlation to Doppler results. In conclusion, absence of physiological changes is associated with abnormal uterine artery Doppler flow and pregnancy complications. However, there is a gradient in the severity of uteroplacental vascular pathology and the correlation with pregnancy complications is not as strong as previously thought. There is also a significant degree of uteroplacental vascular pathology in normal pregnancies with normal uterine artery Doppler flow. This variation may be partly due to sampling error, as a typical biopsy contains only one or two spiral arteries. We hypothesize that additional factors might be necessary to induce the clinical syndrome of pre-eclampsia.

A refined protocol for conducting the low pH 6.7 Syrian hamster embryo (SHE) cell transformation assay.

The Syrian hamster embryo (SHE) cell transformation assay evaluates the potential of chemicals to induce morphological transformation in karyotypically normal primary cells. Induction of transformation has been shown to correlate well with the carcinogenicity of many compounds in the rodent bioassay. Historically the assay has not received wide-spread use due to technical difficulty. An improved protocol for a low pH 6.7 assay was developed by LeBoeuf et al. [R.A. LeBoeuf, G.A. Kerckaert, M.J. Aardema, D.P. Gibson, R. Brauninger, R.J. Isfort, Mutat. Res., 356 (1996) 85-127], that greatly reduced many of the technical difficulties associated with the SHE assay. The purpose of this paper is to describe the most current execution of the pH 6.70 protocol including protocol refinements made since the publication of a comprehensive protocol for this assay in Kerckaert et al. [G.A. Kerckaert, R.J. Isfort, G.J. Carr, M.J. Aardema, Mutat. Res., 356 (1996) 65-84].

Doppler ultrasound screening predicts recurrence of poor pregnancy outcome in subsequent pregnancies, but not the recurrence of PIH or preeclampsia.

OBJECTIVE: To assess the role of Doppler uterine artery screening in the prediction of recurring hypertensive disorders in a high-risk population. METHODS: Ninety-four women with a history of hypertensive disorders in previous pregnancies underwent ultrasound color Doppler to analyze blood flow in the uterine arteries at 21-22 weeks of gestation. We evaluated the performance of the Pulsatility Index (PI) as well as the diastolic notch to predict recurring hypertensive disorders. Outcome measures were the recurrence of hypertensive disorders, and poor pregnancy outcome due to intrauterine death growth retardation, intrauterine death, placental abruption, hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome, eclampsia, or premature birth. Onset of symptoms was before 35 weeks in all cases of poor pregnancy outcome. RESULTS: Doppler flow recordings were obtained from a well-defined location in both uterine arteries. The predictive value of the uterine artery PI for recurring hypertensive disease was poor and not significant; interestingly, however, the predictive values for poor pregnancy outcome were good (sensitivity 83%, specificity 71%, p < 0.001). The PI also provides a good test for intrauterine growth retardation (sensitivity 80%, specificity 69%, p < 0.01). The "diastolic notch" did not perform as well as the PI. CONCLUSIONS: Uterine artery screening did significantly predict the recurrence of poor pregnancy outcome due to hypertensive complications in this high-risk group. In contrast, gestational hypertension and preeclampsia with normal pregnancy outcome were not significantly predicted by uterine artery screening.

Report from the In Vitro Micronucleus Assay Working Group.

At the Washington International Workshop on Genotoxicity Test Procedures (March 25-26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA-specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one-third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis-block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 microg/ml [lymphocytes cultured from whole blood cells] and 3-6 microg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages.

Concepts of threshold in mutagenesis and carcinogenesis.

Although the existence of a threshold in the dose effect relationship is well documented for many, if not most, types of toxicological effects the existence of a threshold for the mutagenic effects of ionising radiation and of certain chemicals has been questioned since the middle of the century and only recently the question of thresholds for radiation and chemical carcinogenesis has been addressed. The essential facts for the interpretation of threshold dose-response curves are common to all type of effects and are: (i) the number and the identity of the target; (ii) the type and sensitivity of the endpoint used to quantify the effect. We therefore will first try to model the type of interactions which may be expected between a mutagen and its target and define from this whether a threshold dose-effect can be expected; in a second step the concept will be extended to heritable mutations and carcinogenesis.

Thresholds in genotoxicity responses.

It has been commonly accepted that risk assessments of genotoxic chemicals are based on linear extrapolation methods. However, there is substantial evidence that some chemicals may be genotoxic only at high doses by mechanisms that do not occur at low doses, or only under specific conditions in genotoxicity assays, but are inactive at concentrations within the range of human exposure levels. There are a variety of possible mechanisms of thresholded genotoxicity, including disruption of cell division and chromosome segregation, inhibition of DNA synthesis, overloading of oxidative defence mechanisms, metabolism or plasma binding capacity, disturbances of metal homeostasis, cytotoxicity and physiological perturbations in in vivo assays. The degrees of evidence supporting the proposed mechanisms are variable and not all are sufficiently robust to be universally accepted as yet by the scientific community. However, a survey of industrial companies indicated that data have been accepted by some regulatory authorities indicating thresholds contributing to genotoxicity responses.

Quantification of the diastolic notch in Doppler ultrasound screening of uterine arteries.

OBJECTIVE: To investigate a new method of quantification of the diastolic notch of the flow velocity waveforms of uterine arteries in the prediction of hypertensive disorders of pregnancy. METHODS: Pulsed-wave Doppler was used to obtain flow velocity waveforms (FVWs) from the uterine arteries at 21-22 weeks of gestation from 531 nulliparous women and 94 multiparous women at high risk. From the FVWs, both the pulsatility index (PI) and the notch index (NI) were calculated and the predictive values for both indices were compared using logistic regression analysis for mild and severe early onset hypertensive pregnancy complications. RESULTS: Both the PI and the NI were poor predictors for mild gestational hypertension and pre-eclampsia; predictive values for severe early onset disease, however, were much better. Logistic regression analysis showed the NI has no additional value compared with the PI in the prediction of either mild or severe disease. CONCLUSIONS: The NI offers the possibility to quantify the diastolic notch in uterine artery analysis. Compared to the PI, this does not lead to better predictive values for hypertensive disorders of pregnancy.

Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing.

Since the p53 gene function is critical to how a cell responds to DNA damage, we investigated the p53 status in Chinese hamster cell lines commonly used in genotoxicity tests for cytogenetic damage around the world. These included: Chinese hamster ovary K1 (CHO-K1), Chinese hamster ovary WBL (CHO-WBL), and Chinese hamster lung (CHL) cells. The results of DNA sequencing, protein analysis, and cell cycle analysis demonstrate that the CHO-K1 and CHO-WBL cell lines have mutant p53 sequence [a mutation in codon 211 in exon 6 resulting in a change from Thr (ACA) to Lys (AAA)], mutant protein (high spontaneous levels that are non-inducible after X-irradiation), and mutant function (lack of G1 checkpoint). Interestingly, the CHL cell line has a completely wild-type p53 DNA sequence. However, the CHL cells have an abnormally high spontaneous level of wild-type p53 protein expression that is not inducible after X-irradiation, yet there is some evidence of G1 delay after irradiation. The protein data suggests that p53 in CHL cells is not being regulated normally, and thus is probably not functioning normally. The mechanism leading to this abnormal regulation of p53 in CHL cells clearly does not involve mutation in the p53 gene. Overall, the CHL cell line may be similar to the CHO cell lines, in that they all appear to have abnormal p53 function. Further work is needed to determine whether the presence of spontaneously high levels of wild-type p53 in CHL cells results in a difference in response to DNA damage (quantitatively or qualitatively) compared to the p53 mutant CHO cell lines.

Comparative genotoxicity of quinolone and quinolonyl-lactam antibacterials in the in vitro micronucleus assay in Chinese hamster ovary cells.

The in vitro micronucleus assay is gaining increased attention as a potential alternative to the standard in vitro metaphase analysis assay. In particular, the in vitro micronucleus assay has been proposed as a useful method for chemicals that induce both structural and numerical chromosome alterations, such as DNA gyrase/topoisomerase inhibitors. In this study, we compared the micronucleus-inducing activity of quinolonyl-lactam antibacterials that inhibit DNA-gyrase and bind to penicillin-binding proteins relative to the activity of structurally related quinolone antibacterials that also inhibit DNA-gyrase. All of the quinolones that were structurally related to the quinolonyl-lactams were cytotoxic and induced large increases in the frequency of micronucleated binucleated cells (MNBC) at concentrations between 0.02 and 0.16 mM. These changes were larger than those seen with the commercial quinolones, ciprofloxacin (cytotoxic at > or = 0.57 mM and MNBC at > or = 0.3 mM) and nalidixic acid (cytotoxic at 1.8 mM and no MNBC up to this dose). In contrast, the quinolonyl-lactams were not cytotoxic up to 1.0 mM concentrations and induced either no MNBC or a low frequency of MNBC at higher concentrations compared to the quinolones. Quinolonyl-lactams appear to be less cytotoxic and genotoxic than structurally related quinolones. These results add to the growing database on the in vitro micronucleus assay in general, and more specifically to the relatively small database for the in vitro micronucleus assay in Chinese hamster ovary cells.