Derivation of point of departure (PoD) estimates in genetic toxicology studies and their potential applications in risk assessment.

Genetic toxicology data have traditionally been employed for qualitative, rather than quantitative evaluations of hazard. As a continuation of our earlier report that analyzed ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) dose-response data (Gollapudi et al., 2013), here we present analyses of 1-ethyl-1-nitrosourea (ENU) and 1-methyl-1-nitrosourea (MNU) dose-response data and additional approaches for the determination of genetic toxicity point-of-departure (PoD) metrics. We previously described methods to determine the no-observed-genotoxic-effect-level (NOGEL), the breakpoint-dose (BPD; previously named Td), and the benchmark dose (BMD10 ) for genetic toxicity endpoints. In this study we employed those methods, along with a new approach, to determine the non-linear slope-transition-dose (STD), and alternative methods to determine the BPD and BMD, for the analyses of nine ENU and 22 MNU datasets across a range of in vitro and in vivo endpoints. The NOGEL, BMDL10 and BMDL1SD PoD metrics could be readily calculated for most gene mutation and chromosomal damage studies; however, BPDs and STDs could not always be derived due to data limitations and constraints of the underlying statistical methods. The BMDL10 values were often lower than the other PoDs, and the distribution of BMDL10 values produced the lowest median PoD. Our observations indicate that, among the methods investigated in this study, the BMD approach is the preferred PoD for quantitatively describing genetic toxicology data. Once genetic toxicology PoDs are calculated via this approach, they can be used to derive reference doses and margin of exposure values that may be useful for evaluating human risk and regulatory decision making.

Quantitative approaches for assessing dose-response relationships in genetic toxicology studies.

Genetic toxicology studies are required for the safety assessment of chemicals. Data from these studies have historically been interpreted in a qualitative, dichotomous "yes" or "no" manner without analysis of dose-response relationships. This article is based upon the work of an international multi-sector group that examined how quantitative dose-response relationships for in vitro and in vivo genetic toxicology data might be used to improve human risk assessment. The group examined three quantitative approaches for analyzing dose-response curves and deriving point-of-departure (POD) metrics (i.e., the no-observed-genotoxic-effect-level (NOGEL), the threshold effect level (Td), and the benchmark dose (BMD)), using data for the induction of micronuclei and gene mutations by methyl methanesulfonate or ethyl methanesulfonate in vitro and in vivo. These results suggest that the POD descriptors obtained using the different approaches are within the same order of magnitude, with more variability observed for the in vivo assays. The different approaches were found to be complementary as each has advantages and limitations. The results further indicate that the lower confidence limit of a benchmark response rate of 10% (BMDL(10) ) could be considered a satisfactory POD when analyzing genotoxicity data using the BMD approach. The models described permit the identification of POD values that could be combined with mode of action analysis to determine whether exposure(s) below a particular level constitutes a significant human risk. Subsequent analyses will expand the number of substances and endpoints investigated, and continue to evaluate the utility of quantitative approaches for analysis of genetic toxicity dose-response data.

Regulatory aspects of genotoxicity testing: from hazard identification to risk assessment.

This symposium focused on the use of tests for chromosomal damage, and other genotoxicity measures, for detection of potentially harmful chemicals. The speakers discussed the information that has been gained over the last three decades about the use of "short-term tests" for genotoxicity in cultured cells and in animals (mainly rodents), and the ongoing debates about the rational use of data from such experimental systems in trying to extrapolate to an understanding of potential human risk. The overall theme was that the field of regulatory toxicology currently is over-reliant on qualitative outcomes of in vitro hazard-screening tests, generally conducted at the maximum achievable exposures, and needs a more realistic approach that incorporates in vivo exposure levels and dose-response information.

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.

Gene expression profiles and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells.

Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P < 0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P < 0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P < 0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.

Immunohematotoxicity studies with combinations of dapsone and zidovudine.

We investigated the immunohematoxicities of the antiparasitic drug dapsone (DDS) and the antiretroviral drug zidovudine (ZDV, AZT) given alone or in combination in BALB/c mice. DDS is used for prophylaxis and treatment of Pneumocystis carinii infection in AIDS patients. We examined the impact of concurrent administration of these drugs on the immune and hematopoietic systems because DDS causes hematotoxicity and ZDV therapy results in bone marrow toxicity. Daily oral administration of DDS at 25 and 50 mg/kg for 28 days caused a slight anemia, marked methemoglobinemia, reticulocytosis, and a moderate leukopenia (P < 0.01 for all parameters) but had no discernible effect on platelet count. In DDS-treated mice, the proliferative response of splenic T cells to concanavalin A was > or = 35% higher than that manifested by splenocytes from vehicle-treated control mice. ZDV at 240 and 480 mg/kg was not immunosuppressive but caused low-grade macrocytic anemia, thrombocytosis, and neutropenia; these effects were drug dose-dependent and statistically significant (P < 0.01). Concurrent administration of DDS and ZDV augmented the severity of ZDV-mediated macrocytic anemia, and 7 of 12 (58%) mice did not survive treatment with the high doses of DDS and ZDV (50 and 480 mg/kg, respectively). On the other hand, co-administration of ZDV mitigated DDS-induced methemoglobinemia and the DDS-associated elevation in lymphoproliferative response. These data suggest interaction between DDS and ZDV in mice and indicate a need for caution in using DDS as long-term therapy in AIDS patients receiving ZDV.

In vitro human tissue models in risk assessment: report of a consensus-building workshop.

Advances in the technology of human cell and tissue culture and the increasing availability of human tissue for laboratory studies have led to the increased use of in vitro human tissue models in toxicology and pharmacodynamics studies and in quantitative modeling of metabolism, pharmacokinetic behavior, and transport. In recognition of the potential importance of such models in toxicological risk assessment, the Society of Toxicology sponsored a workshop to evaluate the current status of human cell and tissue models and to develop consensus recommendations on the use of such models to improve the scientific basis of risk assessment. This report summarizes the evaluation by invited experts and workshop attendees of the current status of such models for prediction of human metabolism and identification of drug-drug interactions, prediction of human toxicities, and quantitative modeling of pharmacokinetic and pharmaco-toxicodynamic behavior. Consensus recommendations for the application and improvement of current models are presented.

Strategies and testing methods for identifying mutagenic risks.

The evolution of testing strategies and methods for identification of mutagenic agents is discussed, beginning with the concern over potential health and population effects of chemical mutagens in the late 1940s that led to the development of regulatory guidelines for mutagenicity testing in the 1970s and 1980s. Efforts to achieve international harmonization of mutagenicity testing guidelines are summarized, and current issues and needs in the field are discussed, including the need for quantitative methods of mutagenic risk assessment, dose-response thresholds, indirect mechanisms of mutagenicity, and the predictivity of mutagenicity assays for carcinogenicity in vivo. Speculation is offered about the future of mutagenicity testing, including possible near-term changes in standard test batteries and the longer-term roles of expression profiling of damage-response genes, in vivo mutagenicity testing methods, and models that better account for differences in metabolism between humans and laboratory model systems.

Micronucleated erythrocyte frequency in peripheral blood of B6C3F(1) mice from short-term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program.

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short-term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short-term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short-term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN-PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30-day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity.

Oral treatment with trimethoprim-sulfamethoxazole and zidovudine suppresses murine accessory cell-dependent immune responses.

Trimethoprim-sulfamethoxazole (TMP-SMX), commonly used for prophylaxis of Pneumocystis carinii pneumonia (PCP) in AIDS patients, often produces a high incidence of treatment-limiting reactions. We investigated the effect of oral administration of TMP-SMX alone or in combination with the antiretroviral drug zidovudine (ZDV) on hematopoiesis and cellular immunity in BALB/c mice. Daily treatment for 28 days with TMP-SMX (160:800 mg/kg) had no effect on hematopoiesis or the ex vivo proliferative response of splenic T lymphocytes to allogeneic tumor cells (EL-4) or to concanavalin A (ConA), or that of splenic B cells to lipopolysaccharide (LPS). ZDV at 240 mg/kg/day was not immunosuppressive but caused a mild macrocytic anemia. Combined treatment produced severe pancytopenia, a significant drop in splenic cellularity, and a 61% decrease in the percentage of splenic macrophages. The percentage of splenic CD3+ lymphocytes increased 150% in the TMP-SMX + ZDV group, but the ratios of T-cell subsets and the frequency of B cells remained unchanged. Combined drug treatment did not impair the proliferative response of B cells to LPS or that of T cells to EL-4 cells. In concert with the reduction in the percentage of macrophages, the proliferative response of T lymphocytes to ConA decreased significantly. Optimal ConA-induced T-cell proliferation requires the participation of accessory cells (AC) (e.g., macrophages); EL-4 cells are able to function as AC. These data indicate that ZDV synergizes with TMP-SMX, causing severe hematotoxicity and suppressing AC-dependent immune function, and suggest that this therapeutic regimen may contribute to the immune deterioration in AIDS patients.

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Pyrimethamine impairs host resistance to infection with Listeria monocytogenes in BALB/c mice.

Increased mortality has been observed when HIV-infected patients were treated with pyrimethamine (Pyr) as prophylaxis for toxoplasmic encephalitis, suggesting that Pyr might possess immunosuppressive activity. To analyze this in an animal model, immune function was assessed in BALB/c mice using a battery of in vivo and ex vivo assays and an in vivo model of host resistance to Listeria monocytogenes infection. Treatment for 30 days with 60 mg/kg Pyr decreased circulating white blood cell and lymphocyte counts but not neutrophil, red blood cell, or platelet counts or hemoglobin levels. Splenic B cell percentages and lipopolysaccharide-induced B cell proliferation decreased significantly after treatment with 60 mg/kg Pyr, as did levels of anti-keyhole limpet hemocyanin (KLH) IgM in serum 7 days after immunization with KLH. Anti-KLH IgG levels 14 days after immunization were not affected. Percentages of splenic T cells and macrophages and T cell proliferation in the presence of concanavalin A or allogeneic cells were not decreased by Pyr treatment. An ex vivo assay of T-cell-mediated cytotoxicity was also unaffected. When host resistance to L. monocytogenes infection was assessed, dramatic increases in mortality were observed in Pyr-treated compared to control mice. Increased numbers of L. monocytogenes organisms were observed in liver and spleen of Pyr-treated mice, compared to controls. The reduction in Listeria resistance, which is T cell mediated, contrasts with the fact that no significant changes in T-cell-mediated immunity were observed. It is possible that Pyr affects parameters of innate immunity, which were not monitored in this study.

'Spontaneous' genetic damage in man: evaluation of interindividual variability, relationship among markers of damage, and influence of nutritional status.

The 'spontaneous' frequency of genetic damage (normal background) and the possible relationship of this damage to nutritional variables in humans were investigated in 22 subjects using several indices of genetic damage. The subjects were chosen, out of 122 initially analyzed, for being at the extremes of the highest and lowest values of one index of genetic damage, the frequency of micronucleated erythrocytes in peripheral blood. This index reflects chromosomal damage and loss in bone marrow erythropoietic cells. The assay for micronuclei is convenient but is restricted to splenectomized individuals because the human spleen removes micronucleated cells. The initial 122 subjects were splenectomized, but all were normal and healthy at the time of this study and none had a previous history of neoplastic disease. Factors investigated were stability of micronucleus frequency as a function of time, correlations among multiple markers of genetic damage, and influence on damage indices of nutritional variables, including blood levels of folate, B12 and antioxidant vitamins. Among different individuals, the range of values was 10-fold or more in the erythrocyte micronucleus, glycophorin A, plasma ascorbate and urinary 8-hydroxydeoxyguanosine (oxo8dG) assays, was approximately 6-fold in the lymphocyte micronucleus assay, and was 2-fold in the lymphocyte sister chromatid exchange (SCE) assay. Red blood cell folate and plasma folate, B12 and alpha-tocopherol values varied by up to 10-fold among individuals. Micronucleus frequencies in erythrocytes and peripheral blood lymphocytes ranged from < 0.3 to 16.9/1000 in mature red blood cells, < 1 to 33/1000 in reticulocytes, and 2.5 to 15/1000 in binucleate lymphocytes. Frequencies of glycophorin A variant erythrocytes ranged from 5.6 to 77.3 x 10(6) N/0 cells and 3.2 to 16.2 x 10(6) N/N cells, and oxo8dG excretion varied from 32 to 397 pmol/kg/day. Although a wide range of values was observed in each genetic endpoint, the extreme values for various endpoints of genetic damage were not observed in the same individuals. The frequency of micronucleated erythrocytes varied over time within individuals and indicated that individuals with the highest levels of damage exhibit greater variability than those with lower levels. In some subjects, frequencies of micronucleated erythrocytes changed dramatically over an interval of 2-3 years: four subjects with initial micronucleated reticulocyte frequencies of 20.4, 5.9, 6.4 and 33/1000 changed to 2.5, 20.5, 18.5 and 12/1000, respectively. Among more than 150 individuals we have studied, including the 64 individuals studied by Everson et al. [(1988) J. Natl. Cancer Inst., 80, 525-529] and Smith et al. [(1990) Cancer Res., 50, 5049-5054], the seven individuals with the highest observed frequencies of micronucleated erythrocytes all had exceptionally low values of plasma folate, red cell folate, or plasma B12, suggesting that folate and B12 status are the major determinants of the types of damage that lead to spontaneous micronucleus formation in erythrocytic cells.

Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage.

Folate deficiency causes massive incorporation of uracil into human DNA (4 million per cell) and chromosome breaks. The likely mechanism is the deficient methylation of dUMP to dTMP and subsequent incorporation of uracil into DNA by DNA polymerase. During repair of uracil in DNA, transient nicks are formed; two opposing nicks could lead to chromosome breaks. Both high DNA uracil levels and elevated micronucleus frequency (a measure of chromosome breaks) are reversed by folate administration. A significant proportion of the U.S. population has low folate levels, in the range associated with elevated uracil misincorporation and chromosome breaks. Such breaks could contribute to the increased risk of cancer and cognitive defects associated with folate deficiency in humans.

Determination of tissue distribution of an intramuscular plasmid vaccine using PCR and in situ DNA hybridization.

The increasing use of nucleic acid-based therapeutics has created a need for new methods of determining tissue distribution and levels. Radiolabel methods may not always be appropriate because nucleic acids are easily degraded. Quantitation using the polymerase chain reaction (PCR) has the advantage that only continuous stretches of DNA will be amplified. In situ hybridization allows detection of specific sequences in histological preparations. We have used quantitative PCR and in situ hybridization techniques to study the pharmacokinetics and distribution of PGagPol (a potential anti-HIV plasmid vaccine) in rabbits. Samples were obtained 4 hr, 24 hr, 7 days, and 28 days after intramuscular injection of 100 micrograms or 400 micrograms of plasmid. A simplified procedure for collecting and processing tissues for PCR that minimizes the risk of contamination was developed. Using PCR, plasmid was found principally in the skin and muscle of the injection site and in blood plasma. At 4 hr after dosing with 400 micrograms, the plasmid was detected at the injection site with mean copy numbers of 10(6) (in muscle) and 4 x 10(4) (in skin) per microgram of tissue. Plasmid copy number declined rapidly in muscle during the first 24 hr and was undetectable at 7 and 28 days after injection. The decline was slower in the skin, and the plasmid was still detectable at 28 days. With in situ hybridization, plasmid was detected in muscle, mainly in the perimysium and to a lesser degree in the endomysium and within the muscle fibers. These data indicate that quantitative PCR and in situ hybridization are sensitive methods for examining tissue distribution of DNA used for gene therapy.