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In epidemiological and clinical studies, time-to-event data often violate the assumptions of Cox regression due to the presence of time-dependent covariate effects and unmeasured risk factors. An alternative approach, which does not require proportional hazards, is to use a first hitting time model which treats a subject's health status as a latent stochastic process that fails when it reaches a threshold value. Although more flexible than Cox regression, existing methods do not account for unmeasured covariates in both the initial state and the rate of the process. To address this issue, we propose a Bayesian methodology that models an individual's health status as a Wiener process with subject-specific initial state and drift. Posterior inference proceeds via a Markov chain Monte Carlo methodology with data augmentation steps to sample the final health status of censored observations. We apply our method to data from melanoma patients with nonproportional hazards and find interesting differences from a similar model without random effects. In a simulation study, we show that failure to account for unmeasured covariates can lead to inaccurate estimates of survival probabilities.

A case-control study of lung cancer mortality in U.S. railroad workers in jobs with and without diesel exhaust exposure is reanalyzed using a new threshold regression methodology. The study included 1256 workers who died of lung cancer and 2385 controls who died primarily of circulatory system diseases. Diesel exhaust exposure was assessed using railroad job history from the US Railroad Retirement Board and an industrial hygiene survey. Smoking habits were available from next-of-kin and potential asbestos exposure was assessed by job history review. The new analysis reassesses lung cancer mortality and examines circulatory system disease mortality. Jobs with regular exposure to diesel exhaust had a survival pattern characterized by an initial delay in mortality, followed by a rapid deterioration of health prior to death. The pattern is seen in subjects dying of lung cancer, circulatory system diseases, and other causes. The unique pattern is illustrated using a new type of Kaplan-Meier survival plot in which the time scale represents a measure of disease progression rather than calendar time. The disease progression scale accounts for a healthy-worker effect when describing the effects of cumulative exposures on mortality.

Babies born live under 2,500 g or with a gestational age under 37 weeks are often inadequately developed and have elevated risks of infant mortality, congenital malformations, mental retardation, and other physical and neurological impairments. In this paper, we model birth weight as a first hitting time (FHT) of a birthing boundary in a Wiener process representing fetal development. We associate the parameters of the process and boundary with covariates describing maternal characteristics and the birthing environment using a relatively new regression methodology called threshold regression. Two FHT models for birth weight are developed. One is a mixture model and the other a competing risks model. These models are tested in a case demonstration using a 4%-systematic sample of the more than four million live births in the United States in 2002. An extensive data set for these births was provided by the National Center for Health Statistics. The focus of this paper is on the conceptual framework, models and methodology. A full empirical study is deferred to a later occasion.

This article proposes nonparametric inference procedures for analyzing microarray gene expression data that are reliable, robust, and simple to implement. They are conceptually transparent and require no special-purpose software. The analysis begins by normalizing gene expression data in a unique way. The resulting adjusted observations consist of gene-treatment interaction terms (representing differential expression) and error terms. The error terms are considered to be exchangeable, which is the only substantial assumption. Thus, under a family null hypothesis of no differential expression, the adjusted observations are exchangeable and all permutations of the observations are equally probable. The investigator may use the adjusted observations directly in a distribution-free test method or use their ranks in a rank-based method, where the ranking is taken over the whole data set. For the latter, the essential steps are as follows: (1) Calculate a Wilcoxon rank-sum difference or a corresponding Kruskal-Wallis rank statistic for each gene. (2) Randomly permute the observations and repeat the previous step. (3) Independently repeat the random permutation a suitable number of times. Under the exchangeability assumption, the permutation statistics are independent random draws from a null cumulative distribution function (c.d.f) approximated by the empirical c.d.f Reference to the empirical c.d.f tells if the test statistic for a gene is outlying and, hence, shows differential expression. This feature is judged by using an appropriate rejection region or computing a p-value for each test statistic, taking into account multiple testing. The distribution-free analog of the rank-based approach is also available and has parallel steps which are described in the article. The proposed nonparametric analysis tends to give good results with no additional refinement, although a few refinements are presented that may interest some investigators. The implementation is illustrated with a case application involving differential gene expression in wild-type and knockout mice of an E. coli lipopolysaccharide (LPS) endotoxin treatment, relative to a baseline untreated condition.

This article examines the application of a first hitting time (FHT) model, using an operational time scale, to assess mortality risk differentials of the work environment. A major case application is presented that applies the model to three job categories of railroad workers. The data set involves a study of more than 50 000 workers with mortality assessed from 1959 to 1996. Lung cancer mortality was assessed because of a suspected link to diesel exhaust exposure. Based on a model that stipulates that death occurs when the disease state of a subject first hits a threshold value, the FHT model provides insights into factors influencing disease progression. In this application, in particular, the findings suggest that a job category in 1959 alters the risk of death from lung cancer.

There is considerable scientific interest in knowing the probability that a site-specific transcription factor will bind to a given DNA sequence. Microarray methods provide an effective means for assessing the binding affinities of a large number of DNA sequences as demonstrated by Bulyk et al. (2001, Proceedings of the National Academy of Sciences, USA 98, 7158-7163) in their study of the DNA-binding specificities of Zif268 zinc fingers using microarray technology. In a follow-up investigation, Bulyk, Johnson, and Church (2002, Nucleic Acid Research 30, 1255-1261) studied the interdependence of nucleotides on the binding affinities of transcription proteins. Our article is motivated by this pair of studies. We present a general statistical methodology for analyzing microarray intensity measurements reflecting DNA-protein interactions. The log probability of a protein binding to a DNA sequence on an array is modeled using a linear ANOVA model. This model is convenient because it employs familiar statistical concepts and procedures and also because it is effective for investigating the probability structure of the binding mechanism.

Serial dilution assays are widely employed for estimating substance concentrations and minimum inhibitory concentrations. The Poisson-Bernoulli model for such assays is appropriate for count data but not for continuous measurements that are encountered in applications involving substance concentrations. This paper presents practical inference methods based on a log-normal model and illustrates these methods using a case application involving bacterial toxins.

Many models have been proposed that relate failure times and stochastic time-varying covariates. In some of these models, failure occurs when a particular observable marker crosses a threshold level. We are interested in the more difficult, and often more realistic, situation where failure is not related deterministically to an observable marker. In this case, joint models for marker evolution and failure tend to lead to complicated calculations for characteristics such as the marginal distribution of failure time or the joint distribution of failure time and marker value at failure. This paper presents a model based on a bivariate Wiener process in which one component represents the marker and the second, which is latent (unobservable), determines the failure time. In particular, failure occurs when the latent component crosses a threshold level. The model yields reasonably simple expressions for the characteristics mentioned above and is easy to fit to commonly occurring data that involve the marker value at the censoring time for surviving cases and the marker value and failure time for failing cases. Parametric and predictive inference are discussed, as well as model checking. An extension of the model permits the construction of a composite marker from several candidate markers that may be available. The methodology is demonstrated by a simulated example and a case application.

Count data often show overdispersion compared to the Poisson distribution. Overdispersion is typically modeled by a random effect for the mean, based on the gamma distribution, leading to the negative binomial distribution for the count. This paper considers a larger family of mixture distributions, including the inverse Gaussian mixture distribution. It is demonstrated that it gives a significantly better fit for a data set on the frequency of epileptic seizures. The same approach can be used to generate counting processes from Poisson processes, where the rate or the time is random. A random rate corresponds to variation between patients, whereas a random time corresponds to variation within patients.

Engineering degradation tests allow industry to assess the potential life span of long-life products that do not fail readily under accelerated conditions in life tests. A general statistical model is presented here for performance degradation of an item of equipment. The degradation process in the model is taken to be a Wiener diffusion process with a time scale transformation. The model incorporates Arrhenius extrapolation for high stress testing. The lifetime of an item is defined as the time until performance deteriorates to a specified failure threshold. The model can be used to predict the lifetime of an item or the extent of degradation of an item at a specified future time. Inference methods for the model parameters, based on accelerated degradation test data, are presented. The model and inference methods are illustrated with a case application involving self-regulating heating cables. The paper also discusses a number of practical issues encountered in applications.

The history of X rays in biological research began almost simultaneously with Roentgen's discovery of his new rays. The history may be unique because of the remarkable collaboration of physicists, chemists, biologists and clinicians--collaborations which have produced and are continuing to produce major contributions to both biological and medical science. These contributions include the use of X rays to investigate molecular structure and function, the first demonstration of induced mutagenesis, the delineation of the cell cycle, the initiation of in vitro and in vivo cloning of mammalian cells, and original studies in DNA repair. The following is a personal overview of the history of some of these developments and their relationship to areas of current biological research. In each case an attempt has been made to trace developments from an early observation or observations to the current day. The history has been divided into two segments, each of approximately 50 years. This division seems appropriate because the separation occurs at approximately the same time as developments which were to play a major role in determining the future of radiation research.

It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene.

We have previously reported the isolation of CHO cell lines resistant to mitomycin C under aerobic conditions of drug exposure. Here it is reported that these cell lines have the same response to mitomycin C under hypoxic conditions as do controls. The cells are shown to have lower levels of DT-diaphorase activity than controls, but similar levels of activity of NADPH:cytochrome c reductase, another enzyme involved in the metabolism of mitomycin C. Evidence for molecular defects in the DT-diaphorase gene or gene transcript is presented for the deficient cell lines. The consequences of this DT-diaphorase deficiency is further explored by testing the toxicity of menadione, an established enzyme substrate. The isolation of CHO cell lines deficient in DT-diaphorase activity and resistant to mitomycin C under aerobic but not hypoxic conditions suggests that mitomycin C reduction by this enzyme has a significant impact on cytotoxicity under aerobic but not hypoxic conditions. Similarly, DT-diaphorase metabolism of menadione does not appear to have a significant impact on cytotoxicity in CHO cells.

The antitumor acridine derivative 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) is at present being evaluated in Phase 2 clinical trials. Exposure of exponential-phase Chinese hamster V79-171b cells to physiologically attainable concentrations of m-AMSA for 60 min generates survival curves with little or no threshold region and an initial D0 of 0.245 +/- 0.019 (S.D.) microM under standard conditions of assay. A minor subpopulation of apparently drug-resistant cells is revealed at low survivals, but these cells on culturing do not display a stable drug-resistant phenotype. m-AMSA survival curves for Chinese hamster ovary cells display features similar to the above. Sensitivity of V79-171b cells to m-AMSA is maximal near pH 7.2 and is markedly reduced by the presence of fetal calf serum. Hypoxia has little effect on the toxicity of m-AMSA, and repair of potentially lethal damage has not been observed after treatment with this agent. Noncycling plateau-phase V79-171b or Chinese hamster ovary cells are markedly less sensitive to m-AMSA than are early log-phase cells. This resistance to m-AMSA appears to be related to the slowly cycling or noncycling status of plateau-phase cells, suggesting that the cytokinetic character of cell populations in vivo will be a major determinant of their sensitivity to this drug. However, the increase in resistance to m-AMSA during growth into plateau-phase appears to commence well before departure from exponential growth can be detected and may thus be a consequence of metabolic changes more subtle than the transition from a cycling to a noncycling state.

In aqueous solution, in the presence of ammonium chloride, N1-substituted 2-nitroimidazoles are readily reduced to the corresponding hydroxylamines. In air, under neutral conditions, analogous to the reactions of aromatic hydroxylamines, 2-hydroxylaminoimidazoles are converted to the azoxy derivatives via a base-catalyzed condensation reaction between the hydroxylamine and its oxidation product, the nitroso derivative. In nitrogen, rearrangement to form the 2-amino-4(5)hydroxyimidazole derivative followed by addition of water across the C4-C5 double bond to yield isomers of a 4,5-dihydro-4,5-dihydroxy derivative appears to be a major reaction. 2-hydroxylaminoimidazoles undergo a complex series of reactions with glutathione. The initial reaction is the formation of a labile conjugate involving an N-S-linkage. Subsequently in the presence of excess GSH, under neutral conditions, two stable conjugates identified as 2-amino-4-S-glutathionyl- and 2-amino-5-S-glutathionyl imidazoles are formed. Nucleophilic attack by GSH on the imidazole ring of a nitrenium ion is postulated as the initial step in the formation of the stable GSH conjugates as well as the 2-amino-4,5-dihydro dihydroxy derivative. The results provide a molecular mechanism for many of the biological effects of N1-substituted 2-nitroimidazoles in hypoxic mammalian cells.

A simple procedure for isolating mutagen-sensitive clones of CHO cells was developed and applied in mutant hunts in which colonies were screened for hypersensitivity to killing by ultraviolet radiation (UV, ethyl methanesulfonate (EMS), or mitomycin C (MMC). Each of two UV-sensitive clones studied in detail had a D37 dose of 1.0 J/m2 compared to 7.0 J/m2 for the wild-type cells, and each was shown to have no detectable repair replication following exposure to UV doses of up to 26 J/m2. Although these mutants resemble xeroderma pigmentosum human mutants with respect to their repair defect and cross-sensitivity to the carcinogen 4-nitroquinoline-1-oxide, one of two clones (UV-20) is characterized by extreme hypersensitivity to MMC (80-fold as compared to the wild type). Clones having hypersensitivity to alkylating agents, but not UV, were obtained using MMC and EMS. In the latter case the two clones had significantly increased sensitivity to the killing action of 60Co gamma-rays.

Nitroimidazoles, under hypoxic conditions, undergo reduction reactions producing a variety of reactive species; at the same time, they exert a variety of biological effects. The purpose of the present study was to investigate the reduction chemistry of 2-nitroimidazoles, the reaction of the reduced metabolites with nucleic acid constituents and the possible biological significance of these reactions. Earlier studies had demonstrated that, following reduction, 2-nitroimidazoles react with guanine derivatives to produce identical to that seen on reaction of glyoxal with the same guanine derivatives. We report here the identification of this product in the nucleic acid of cells exposed to 2-nitroimidazoles under hypoxic conditions. Using glyoxal as a model compound, we also demonstrate that cellular formation of such a product could account for a number of the biological properties of 2-nitroimidazoles under hypoxic conditions.

Recently, two human DNA-repair genes have been cloned which complement the defects in complementation groups 1 and 2 of the CHO mutants which are sensitive to ultraviolet light and deficient in the incision step of excision repair. Here we report human gene transfer-mediated complementation of a group 4 CHO mutant sensitive to ultraviolet light and mitomycin C (MMC). The transfectants generated by transfecting human DNA into the repair-deficient cell line demonstrate the repair-proficient phenotype, as they have wild-type levels of resistance to UV light and MMC and are competent in performing the incision step of excision repair in response to UV irradiation. 3 of the 8 transfectants isolated display no detectable human repetitive sequences, while the other 5 contain varying amounts of human repetitive DNA. As the evidence suggests that all of the transfectants are repair-proficient as a result of the uptake of human DNA, we conclude that the human gene that complements the repair defect in group 4 CHO mutants contains no highly abundant human repetitive sequences. This imposes the necessity of developing cloning strategies involving the identification of sequences that flank the gene.

Reaction between GSH and the hydroxylamine derivative of SR-2508 results in the formation of two stable conjugates identified as 2-amino-4-S-glutathionyl and 2-amino-5-S-glutathionyl imidazoles. These stable conjugates are apparently formed from a reactive derivative of the hydroxylamine that is sufficiently stable to be isolated after HPLC separation. The physical and chemical properties of this derivative are consistent with it being a GSH conjugate in which the glutathionyl residue is attached to the 2-amino nitrogen of the imidazole moiety through sulphur. With excess GSH, under physiological conditions, it forms a mixture of the two stable GSH conjugates. In CHO cells exposed to SR-2508 under hypoxic conditions, this unstable GSH conjugate has been detected and suggests the possibility of GSH functioning as a carrier of a toxic metabolite of 2-nitroimidazoles under certain conditions.

RSU-1069 combines an aziridine function with a 2-nitroimidazole and has been reported to exhibit extraordinary radiosensitization both in vitro and in vivo. Such sensitization appears to be at variance with the electron affinity of the compound. In addition, recent experiments suggest that the compound is highly toxic to hypoxic tumor cells in vivo. On the assumption that the observed radiosensitizing ability may be a manifestation of toxicity and because of the high in vivo toxicity, we have investigated aerobic and hypoxic toxicity, both in wild type CHO cells and in mutants sensitive to a variety of DNA damaging agents. With wild type cells under aerobic conditions, the compound is approximately 50 times as toxic as misonidazole and under hypoxic conditions, approximately 250 times as toxic. The ratio of hypoxic to aerobic toxicity is approximately 80 times. Under aerobic conditions, repair-deficient mutants are 10 times as sensitive to RSU-1069 as wild type cells and approximately 100 times as sensitive under hypoxic conditions. The ratio of hypoxic to aerobic toxicity for the mutant cells is approximately 900. Based on these observations, we suggest that under aerobic conditions the aziridine function is primarily responsible for toxicity, whereas, under hypoxic conditions, the aziridine moiety combined with a reduced 2-nitroimidazole moiety produces a bifunctional agent.

Misonidazole, after reduction to the hydroxylamine derivative, reacts with glutathione (GSH) under physiological conditions. The reaction product has been identified as a mixture of two isomeric conjugates. When water soluble extracts of CHO cells exposed to misonidazole under hypoxic conditions are subjected to HPLC analysis, misonidazole derivatives, having the same chromatographic properties as the GSH-MISO conjugates, were detected. The identity of the synthetic and cellular products was further confirmed by identification of the amine derivative of misonidazole after desulfurization with Raney Nickel. When CHO cells were incubated with misonidazole in the presence of added GSH, a substantial increase in the amount of the conjugate was detected. When extracts of CHO cells exposed to misonidazole under hypoxia were subsequently exposed to GSH, an increased formation of the conjugate was observed. A rearrangement product of the hydroxylamine derivative of misonidazole is postulated as the reactive intermediate responsible for the formation of the conjugate.

[14C]Misonidazole was reduced with zinc dust in aqueous solution in the presence of ammonium chloride. When the reduction mixture was allowed to react with calf thymus DNA and the reaction mixture was dialyzed, radioactivity was detected in the DNA fraction. Bovine albumin, after reaction with the reduction mixture and subsequent Bio-Gel P-2 column chromatography, was also found to be radioactive. The hydroxylamine derivative of misonidazole was detected in the reduction mixture. It is postulated that binding of nitroreduction products of misonidazole to cellular macromolecules is a probable mechanism for the cytotoxicity of misonidazole.

The cytotoxic activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), a novel acridine derivative with clinical antitumor activity, has been examined in multicellular spheroids grown from Chinese hamster V79-171b cells. m-AMSA is much less effective against cells within these tumor-like structures than it is against exponential-phase V79-171b cells in monolayer cultures, the initial D0 of the survival curve for the latter being approximately 10-fold lower than for the former following a 60-min exposure to the drug. The resistance of spheroid cells to m-AMSA appears to be at least partially a result of the noncycling or slowly cycling state of the majority of these cells, although they are more sensitive than cells in plateau-phase monolayers. A further component of resistance in spheroids requires the presence of an intact spheroid structure and may be due to drug transport limitations. The use of sequential trypsinization techniques to recover cells at varying depths within spheroids demonstrates that a 60-min m-AMSA treatment preferentially kills cells nearest the spheroid surface, suggesting that tumor cells at a distance from the vasculature may limit the efficacy of m-AMSA in vivo.

Although it has long been evident that the response of eukaryotes to DNA damaging agents is determined by the effectiveness of a variety of DNA repair systems, there is little detailed knowledge of the nature of these systems or the genes which control them. In humans, a number of hereditary conditions, including xeroderma pigmentosum, ataxia telangiectasia and Fanconi's anaemia, exhibit increased sensitivity to a variety of DNA damaging agents and a predisposition to cancer, suggesting a defect in some aspect of DNA repair. This report describes the identification of a human DNA repair gene following DNA-mediated gene transfer into Chinese hamster ovary (CHO) mutant cells, that like xeroderma pigmentosum cells, are sensitive to a variety of DNA damaging agents and are defective in the initial incision step of DNA repair. The resulting transformants exhibit normal resistance to DNA damaging agents and independent transformants demonstrate a common set of human DNA sequences associated with a human DNA repair gene. These observations provide the basis for the isolation and characterization of the human genes responsible for DNA repair.

Chemical studies have indicated that, following reduction of misonidazole to the hydroxylamine derivative, reaction with guanosine leads to the formation of a 2-carbon addition product of guanosine. In this study, the formation of the guanosine product is used to detect the presence of a reactive metabolite of misonidazole in the urine of patients treated with misonidazole. Urine samples were incubated with [14C]guanosine and the guanosine product was separated by HPLC analysis. The quantities of product vary as much as 10-fold from patient to patient and it is suggested that the assay might be useful as a predictor of patients susceptible to the development of peripheral neuropathy or other effects of misonidazole.

Reduction of the nitro group occurred when [14C]misonidazole was treated with zinc dust in aqueous solution in the presence of ammonium chloride. When the reduction mixture was allowed to react with calf thymus DNA or bovine albumin, radioactivity was bound to both DNA and protein. Under the same conditions, misonidazole did not bind to these macromolecules. Analysis of the reduction mixture indicated that the hydroxylamine, amine, and hydrazo derivatives of mizonidazole were the major products. In a number of tissues of C3H mice after administration of [14C]misonidazole, radioactivity was detected in the DNA, RNA, and protein fractions. Similar results were also obtained with Chinese hamster ovary cells incubated with the drug in the absence of oxygen. It is postulated that nitroreduction and binding of the nitroreduction products to macromolecules is a probable mechanism for the mutagenic and cytotoxic properties of misonidazole.

The mortality component of contemporary health indexes is discussed. Since these indexes reduce to mortality indexes when only life and death states enter the analysis, they share the conceptual weaknesses of mortality indexes. Also, they do not incorporate consumption variables explicity and therefore provide no structure for relating health status and living standard. Some attention is devoted to methodological problems of assessing survival probabilities, either from survey or experimental data or from beliefs of experts or individuals who are affected directly. The final section deals with individual preferences for survival lotteries. Conceptual weaknesses of common indexes are discussed, several canonical models for survival preferences are presented, the interdependence of individual utilities is discussed, and methods for eliciting individual survival preferences are considered, along with some illustrative empirical results.

The effect of methotrexate on the growth of Chinese hamster cells was examined under a variety of conditions. The experiments suggest that the important biological effects of methotrexate are the result of direct inhibition of thymidylate synthetase and one or both of the folate-dependent enzymes involved in a purine biosynthesis. In addition, analysis of the distribution of intracellular folate derivatives following methotrexate treatment gives no indication of accumulation of dihydrofolate, and accumulation that would be expected if inhibition of dihydrofolate reductase were the principal site of methotrexate action.

Misonidazole is readily reduced by zinc dust in aqueous solution in the presence of ammonium chloride. High pressure liquid chromatographic (HPLC) separation of the reduction mixture revealed the presence of three products. These were identified as the hydroxylamine, amine and the hydrazo derivative of misonidazole. There is evidence that the azoxy derivative was an intermediate in the reduction process. When the reduction was carried out in dilute solution (0.1 mg/ml), the hydroxylamine was the only product. In concentrated solution (20 mg/ml), the hydrazo derivative was the major product. When misonidazole was reduced with hydrogen using palladium as catalyst, the amine was the only detectable product. Of the three products, only the hydroxylamine was found to bind covalently to bovine albumin. In Chinese hamster ovary (CHO) cells under hypoxic conditions the amine was confirmed as one of the metabolites. There was no evidence for the presence of detectable amounts of the hydroxylamine in the cell extracts. These studies suggest that the hydroxylamine is probably the reactive reduction metabolite responsible for the in vivo and in vitro binding of misonidazole to cellular macromolecules.

Genetic mapping studies in bacterial, lower eukaryotic, and mammalian systems have demonstrated that the enzyme or enzyme complex involved in the initial incision step of the DNA excision repair pathway is coded for by more than one genetic locus. This paper reports the results of complementation studies that were performed with a number of DNA repair deficient Chinese hamster ovary cell lines. Complementation abilities were measured by comparing the survival of selected mutant X mutant hybrids with that of the tetrapoloid wild type after exposure to a number of physical and chemical agents. In all cases studied, hybrids formed from two different mutant lines showed resistance similar to that of the wild-type line. These results not only demonstrate that the mutant lines were in different complementation groups, but that complementation of a DNA-repair defect associated with one particular agent can complement the defect for another DNA-damaging agent.

Mitomycin C (MMC), a bifunctional alkylating agent, requires metabolic reduction to become biologically active. We have identified a series of genetically related Chinese hamster ovary cell lines which span approximately three orders of magnitude in the concentration of MMC required for cell killing. Many mechanisms, including drug transport, drug activation, drug detoxification, and the elimination, or repair, of drug-induced lesions, may contribute to the level of drug resistance in cells. By exploring each of the above mechanisms in the various Chinese hamster ovary cell lines, we have been able to classify these cell lines into four categories. Proceeding from least resistant to most resistant to MMC, the cell lines are: (a) proficient in the bioreduction of MMC and deficient in DNA excision repair; (b) deficient in some aspects of MMC bioreduction and deficient in repair; (c) bioreduction and repair proficient; and (d) bioreduction deficient and repair proficient.

Properties of the inverse gaussian distribution are presented with comments on fitting the distribution to lentgh-of-stay data. A conceptual framework for the hospitalization process is described; it suggests that the inverse gaussian distribution has considerable potential as both a descriptive and prescriptive model of length of stay, especially in the setting of psychiatric hospitals.

Misonidazole, after reduction to the hydroxylamine derivative, was found to react with guanosine in aqueous solution at pH 7. The guanosine product was isolated and was assigned a structure having a new 5-membered ring with a -CHOH-CHOH-linkage between the N-1 and N-2 positions of guanine. Removal of the sugar residue from the guanosine product by acid hydrolysis resulted in the corresponding guanine derivative, which was also made by reacting guanine with reduced misonidazole. In aqueous solution at pH 11, the guanine product was quantitatively converted to guanine within 20 min. A number of N-1-substituted 2-nitroimidazoles and 2-nitroimidazole reacted with guanosine in an analogous manner, giving rise to the same product as misonidazole, indicating that the C-4-C-5 fragment from the imidazoles is involved in the modification. Neither misonidazole nor its amine or hydrazo derivatives reacted with guanosine. Reduced misonidazole reacted with N-2-methyl guanosine, whereas with N-1-methyl guanosine a reaction was not detected. The identity of Structure I was confirmed by comparison with an authentic sample of Structure I that was prepared by reacting guanosine with glyoxal. Reactions such as the modification of guanine provide a possible molecular mechanism for the cytotoxic and neurotoxic properties of misonidazole.

The survival of the wild-type parent and two mutant lines of Chinese hamster cells, known to be defective in DNA repair, has been determined as a function of exposure to gamma rays under aerobic and hypoxic conditions. When compared to the wild-type line, one of the mutants selected for sensitivity to ethyl methyl sulfonate (EMS), and known to be defective in the repair of DNA strand breaks, exhibits a markedly enhanced sensitivity to aerobic irradiation but a reduced enhancement to hypoxic irradiation and thus an enhanced oxygen enhancement ratio (OER). In contrast, the other line, known to be defective in the incision step of excision repair, exhibits the reverse pattern of sensitivity and hence a reduced OER. The results are compared to findings in bacterial mutants and cells obtained from ataxia telangiectasia (AT) patients and heterozygotes.

RSU-1069 is a highly effective hypoxic cell cytotoxin in KHT sarcomas treated in vivo. However, relative to the hypoxic cells, the oxic cells in the tumor appear more sensitive to the drug than would have been predicted on the basis of results with CHO (AA8-4) cells treated in vitro with the drug under oxic and hypoxic conditions. To examine possible reasons for this difference, suspensions of KHT cells were prepared from tumors growing in vivo, and treated with RSU-1069 in vitro under oxic or hypoxic conditions. The sensitivity of the KHT cells was similar to that of AA8-4 cells, regardless of whether the cells were obtained from untreated tumors or from tumors given 15 Gy in vivo just prior to the preparation of the cell suspension. We observed, however, that the sensitivity of both AA8-4 cells and KHT cells to drug treatment under hypoxic conditions increased with the density of the cells in the treated suspension. This result suggests the possibility that a diffusible toxic product may be released from cells. Such a product could contribute to the toxicity of the drug for oxic cells in tumors in situ.