James G. Burkhart

An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The literature on the mouse sperm morphology test and on other sperm tests in nonhuman mammals was reviewed (a) to evaluate the relationship of these tests to chemically induced spermatogenic dysfunction, germ-cell mutagenicity, and carcinogenicity, and (b) to make an interspecies comparison to chemicals. A total of 71 papers were reviewed. The mouse sperm morphology test was used to assess the effects of 154 of the 182 chemical agents covered. 4 other murine sperm tests were also used: the induction of acrosomal abnormalities (4 agents), reduction in sperm counts, (6 agents), motility (5 agents), and F1 sperm morphology (7 agents)). In addition, sperm tests for the spermatogenic effects of 35 agents were done in 9 nonmurine mammalian species; these included analyses for sperm count, motility, and morphology, using a large variety of study designs. For the mouse sperm morphology test, 41 agents were judged by the reviewing committee to be positive inducers of sperm-head shape abnormalities, 103 were negative, and 10 were inconclusive. To evaluate the relationship between changes in sperm morphology and germ cell mutagenicity, the effects of 41 agents on mouse sperm shape were compared to available data from 3 different mammalian germ-cell mutational tests (specific locus, heritable translocation, and dominant lethal). The mouse sperm morphology test was found to be highly sensitive to germ-cell mutagens; 100% of the known mutagens were correctly identified as positives in the sperm morphology test. Data are insufficient at present to access the rate of false positives. Although it is biologically unclear why one might expect changes in sperm morphology to be related to carcinogenesis, we found that (a) a positive response in the mouse sperm morphology test is highly specific for carcinogenic potential (100% for the agents surveyed), and (b) overall, only 50% of carcinogens were positive in the test (i.e., sensitivity approximately equal to 50%). Since many carcinogens do not produce abnormally shaped sperm even at lethal doses, negative findings with the sperm test cannot be used to classify agents as noncarcinogens. We conclude that the mouse sperm morphology test has potential use for identifying chemicals that induce spermatogenic dysfunction and perhaps heritable mutations. Insufficient numbers of chemicals agents have been studied by the other sperm tests to permit similar comparisons. A comparison of 25 chemicals tested with sperm counts, motility, and morphology in at least 2 species (including man, mouse and 9 other mammals) demonstrated good agreement in response among species. With further study, interspecies comparisons of chemically induced sperm changes may be useful for predicting and evaluating human effects.

An evaluation of human sperm as indicators of chemically induced alterations of spermatogenic function: A report of the U.S. environmental protection agency Gene-Tox program

To evaluate the utility of sperm tests as indicators of chemical effects on human spermatogenesis, the literature on 4 sperm tests used to assess chemically induced testicular dysfunction was reviewed. The tests surveyed included sperm count, motility, morphology (seminal cytology), and double Y-body (a fluorescence-based test thought to detect Y-chromosomal nondisjunction). There were 132 papers that provided sufficient data for evaluation. These reports encompassed 89 different chemical exposures: 53 were to single agents; 14 to complex mixtures; and 22 to combinations of 2 or more identified agents. Approximately 85% of the exposures were to experimental or therapeutic drugs, 10% were to occupational or environmental agents, and 5% were to drugs for personal use. The most common sperm parameter studied was sperm count (for 87 of the 89 exposures reviewed). Sperm motility was evaluated for 59 exposures, morphology for 44, and double Y-bodies for only 4. The 89 exposures reviewed were grouped into 4 classes: those which adversely effected spermatogenesis, as measured by one or more of the sperm tests (52); those suggestive of improving semen quality (11); those showing inconclusive evidence of adverse effects from exposure (14); and those showing no significant changes (12). Since the reviewed reports had a large variety of study designs, and since every attempt was made to include all reports with interpretable data, these classifications were based on reviewing committee decisions rather than on uniform statistical criteria. This review gives strong evidence that human sperm tests can be used to identify chemicals that affect sperm production, but because of our limited understanding of underlying mechanisms, the extent to which they can detect mutagens, carcinogens or agents that affect fertility remains uncertain. For the very few agents studied with both human and mouse sperm tests, similar test-responses were seen; thus sperm tests in mice and other laboratory mammals may have a potential role in hazard identification. An overall comparison of the 4 human sperm tests suggests that no one test is biologically more responsive than another; all of them may thus be needed when testing for chemically induced changes from agents of unknown activity. This review also gives evidence that sperm tests can be used to assess the extent and the potential reversibility of induced spermatogenic damage. The reviewing committee recommends further studies to determine (a) the dose-response characteristics of the human sperm tests, (b) details of the reversibility of induced changes with time after exposure, (c) the relative responses in the 4 sperm tests in exposed individuals, (d) the mechanism of action, (e) the biological and genetic implications of chemically induced effects, and (f) the comparison of responses among different species for risk assessment. The reviewing committee outlines specific considerations for planning new sperm studies on chemically exposed men.

Hind Limb Malformations in Free-Living Northern Leopard Frogs (Rana pipiens) From Maine, Minnesota, and Vermont Suggest Multiple Etiologies

BACKGROUND Reports of malformed frogs have increased throughout the North American continent in recent years. Most of the observed malformations have involved the hind limbs. The goal of this study was to accurately characterize the hind limb malformations in wild frogs as an important step toward understanding the possible etiologies. METHODS During 1997 and 1998, 182 recently metamorphosed northern leopard frogs (Rana pipiens) were collected from Minnesota, Vermont, and Maine. Malformed hind limbs were present in 157 (86%) of these frogs, which underwent necropsy and radiographic evaluation at the National Wildlife Health Center. These malformations are described in detail and classified into four major categories: (1) no limb (amelia); (2) multiple limbs or limb elements (polymelia, polydactyly, polyphalangy); (3) reduced limb segments or elements (phocomelia, ectromelia, ectrodactyly, and brachydactyly; and (4) distally complete but malformed limb (bone rotations, bridging, skin webbing, and micromelia). RESULTS Amelia and reduced segments and/or elements were the most common finding. Frogs with bilateral hind limb malformations were not common, and in only eight of these 22 frogs were the malformations symmetrical. Malformations of a given type tended to occur in frogs collected from the same site, but the types of malformations varied widely among all three states, and between study sites within Minnesota. CONCLUSIONS Clustering of malformation type suggests that developmental events may produce a variety of phenotypes depending on the timing, sequence, and severity of the environmental insult. Hind limb malformations in free-living frogs transcend current mechanistic explanations of tetrapod limb development.

ENU-induced mutagenesis at a single A: T base pair in transgenic mice containing Φ X174

Transgenic mice containing the bacteriophage phi X174 am3 as a chromosomally integrated and recoverable marker for in vivo mutation have been produced to measure spontaneous and induced substitutions at an A:T base pair among single copies. phi X174 was chosen for its small size (5 kb), unique sequence, and the opportunity to take advantage of previously reported in vitro data on mutation and repair; the am3 site provides sequence specificity in a reversion assay for mutation of an A:T base pair. Inbred C57Bl/6 mice have been made homozygous for approximately 100 copies of the the phage sequence without any apparent detrimental effects on the homozygous individuals. Recoveries of phage from mouse tissues are in the range of 1-5 x 10(7) PFU per micrograms mouse DNA; both recovery and mutation are independent of endogenous CpG methylation. Background mutation frequencies are 2-4 x 10(-7) among phage recovered from liver, brain, spleen, and kidney. Adult mice were treated with 200 mg/kg N-ethyl-N-nitrosourea, and phage were recovered at 2 and 14 days after treatment. At 2 days after treatment we observed a slight increase only among phage isolated from the brain of one mouse out of four. At 14 days after ENU treatment, there were significant increases in mutation frequencies among phage recovered from the liver (6 x) and spleen (10 x). These results demonstrate (1) response of a single A:T base pair to alkylation-induced mutation in a nonexpressed gene, (2) the role of cell proliferation in somatic mutagenesis, and (3) provide a model for a transgenic approach for study of site-specific mutagenesis in vivo in higher eukaryotes.

Effects of pond water, sediment, and sediment extracts from minnesota and vermont, USA, on early development and metamorphosis of xenopus

In recent studies, a high incidence of amphibian mortality and malformation has been reported in the field, suggesting that toxic and/or bioactive agents are present in the environment of the affected amphibians. This study provides evidence for this hypothesis, because it applies to several affected ponds in Minnesota and Vermont, USA. Three developmental bioassays were carried out on samples from three reference and three test sites in Minnesota and one reference and three test sites, in Vermont. The bioassays utilized Xenopus as a model system, measuring altered developmental patterns during the first 4 d of development (frog embryo teratogenesis assay-Xenopus [FETAX]), hind-limb development over a 30-d period, and tail length resorption over a 14-d period. Strong correlations were observed among the results for all three in vitro bioassays, as well as between adverse developmental effects in vitro and in the field.

PROGRESS TOWARD IDENTIFYING CAUSES OF MALDEVELOPMENT INDUCED IN XENOPUS BY POND WATER AND SEDIMENT EXTRACTS FROM MINNESOTA, USA

In previously conducted laboratory studies with the South African clawed frog (Xenopus laevis), pond water and sediment samples collected from various sites in Minnesota, USA, were demonstrated to have the potential to induce a variety of developmental abnormalities, including early embryo-larval maldevelopment, abnormal limb development, and disruption of metamorphosis. The results of exposure of X. laevis to suspect pond water and sediment samples supported the hypothesis that these samples were capable of inducing these abnormalities as the result of either the presence of developmental toxicants or the absence of essential micronutrients. Physicochemical characterization of the causes of abnormal frog embryo-larval and limb development were performed using the frog embryo teratogenesis assay-Xenopus (FETAX). Specific compounds were subsequently identified within the complex mixture fractions and tested by dilution in a control solution and native reference water using both the 4- and 30-d treatment protocols. Results from these studies suggested that a complex mixture of both naturally occurring and man-made compounds was primarily responsible for the effects observed in X. laevis. The potency of several compounds was also enhanced by the site water, thus indicating that the water matrix deserves consideration as a contributing factor for both laboratory and field studies.

Transfer, methylation and spontaneous mutation frequency of ΦX174am3cs70 sequences in medaka (Oryzias latipes) and mummichog (Fundulus heteroclitus): Implications for gene transfer and environmental mutagenesis in aquatic species

Abstract This study describes the production of transgenic medaka ( Oryzias latipes ) and mummichog ( Fundulus heteroclitus ) containing multiple copies of the bacteriophage ΦX174 am3cs70 . This work is an initial approach for measuring mutations in aquatic species using the same gene target sequence in ish and laboratory mammals. The ΦX174 sequence is unique in that there is no detectable homology with chromosomal DNA of medaka, mummichog or mice. The authors have compared cytoplasmic injection of 1–2 cell embryos with linear single copy and catenated constructs of the phage DNA. The catenated construct results in greater efficiency of gene transfer for both species in terms of copies per cell. Analyses of DNA from founder transgenic fish with methylation sensitive (HpaII) and methylation insensitive (MspI) restriction enzyme isoschizmers indicates CpG methylation of the integrated ΦX174 sequence. This study also demonstrates the efficient rescue of live phage from the chromosomal DNA of founder fish in sufficient numbers to determine a spontaneous mutation frequency for reversion of am3 . A pooled sample of 20 μg DNA from four fish yielded 1.09 × 10 7 progeny phage with a spontaneous mutation frequency of 1.83 × 10 −7 . This spontaneous mutation frequency is similar to the spontaneous frequency for the same gene indictor recovered from transgenic mice. These results demonstrate that fish containing multiple copies of ΦX174 can be produced with no obvious detrimental effects and that the overall approach may be useful in basic and applied studies of environmental mutagenesis.

Purification and molecular weight determination of glucose-6-phosphate dehydrogenase and malic enzyme from mouse and Drosophila

Abstract A facile two-step procedure was employed for simultaneous purification of glucose-6-phosphate dehydrogenase and malic enzyme from mouse (strain DBA 2J ) and Drosophila melanogaster. This involved the use of an 8-(6-aminohexyl)-amino-2′,5′-ADP-Sepharsoe affinity column chromatography followed by DEAE-Sephadex chromatography. The native and subunit molecular weights of these two homogeneous enzymes were determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. From this study, it was concluded that the two enzymes are tetrameric and have native molecular weights between 200,000 and 280,000 in both species.

Sensitivity of the ΦX174 am3 allele in relation to the endogenous Hprt gene for detecting mutation in transgenic mice

Transgenic mice have been developed containing multiple, chromosomally integrated copies of the ΦX174 am3 allele that serve as reporters for in vivo mutation at a single A:T basepair. In this study, we examined the relative sensitivity of the am3 transgene for detecting the in vivo mutagenicity of N‐ethyl‐N‐nitrosourea (ENU). Three‐week‐old male ΦX174 mice were treated with 0, 40, and 160 mg/kg of ENU. After 1, 3, 6, and 9 weeks, animals were killed, their spleens removed, and isolated splenocytes were used to measure mutant frequencies (MFs) in both the am3 allele and the endogenous Hprt gene. For animals treated with 40 mg/kg of ENU, the Hprt assay detected an average 22‐fold increase over background, while the am3 MFs averaged threefold above background. With the 160 mg/kg dose, the Hprt assay detected a 54‐fold average increase, while a sixfold average increase above background was found for the transgenic locus. We conclude that the sensitivity of the am3 assay to ENU was compromised by the presence of ex vivo mutations. Adjustment of am3 MFs to exclude these ex vivo mutants could enhance the sensitivity of the assay. Environ. Mol. Mutagen. 32:229–235, 1998

Biochemical and molecular analysis of spontaneous and induced mutations at the mouse Mod-1 locus

We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.