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Background:. Oncoplastic breast reconstruction addresses lumpectomy or partial mastectomy defects after breast conserving surgery. A large subset of breast cancer patients may be candidates for oncoplastic partial breast reconstruction, but are likely underserved in the United States due to multiple factors. In this article, the authors provide an understanding of current trends in the literature and an overview of key areas for improvement in the delivery of breast cancer surgical care. Methods:. A literature search of peer-reviewed articles relating to oncoplastic breast reconstruction was performed. In addition, current US laws relating to breast reconstruction, and communication and content from key surgical societies were reviewed. Key areas of improvement were identified after qualitative review of all sources. Results:. Thirty-four relevant resources were included. There is a large potential population need for oncoplastic breast reconstruction, but no current accurate estimate of patients undergoing this type of reconstruction. There are a multitude of definitions and algorithms attempting to define and organize oncoplastic breast surgery. Legislative initiatives could be improved to prevent coverage gaps. Coding and billing for oncoplastic surgery is not standardized, which complicates both clinical implementation and research efforts. Collaboration between national societies is needed. Conclusions:. Oncoplastic partial breast reconstruction is an important part of the total package of care that can be offered to the breast cancer patient. Improvements in legislation, coding, and billing, and unification of national society communications and initiatives can lead to better surgical outcomes for the breast cancer patient.

Background: Dermatomyositis is an inflammatory myopathy that has an increased risk of malignancy, warranting aggressive health maintenance screenings. Dermatomyositis can rarely present with vulvovaginitis, thus clinical suspicion is important in early diagnosis.Case: We present the case of a 31-year-old woman with a 10-year history of vulvovaginitis as her presenting symptom of dermatomyositis. On further investigation, she had a history of joint pain, muscle pain, fatigue, and leukopenia. On examination, she was found to have a rash and mucopurulent vulvovaginitis. Biopsies from the chest, axilla, and vulva were compatible with dermatomyositis. Evaluation for malignancy was negative, and her symptoms resolved with treatment of the dermatomyositis.Conclusion: Dermatomyositis is an inflammatory disease that can be associated with malignancy. This particular patient presented with vulvovaginitis. This case is important because the diagnosis was delayed. Early recognition is important to evaluate for malignancy. [ABSTRACT FROM AUTHOR]

Objective: In response to previously published findings of methylphenidate-induced chromosomal changes in children, this study was designed to determine whether methylphenidate- or amphetamine-based drugs induce chromosomal damage (structural aberrations, micronuclei, and sister chromatid exchanges) in peripheral blood lymphocytes of children with attention-deficit/hyperactivity disorder after 3 months of continuous treatment.Method: Stimulant drug-naïve subjects, 6 to 12 years of age, in good overall health, and judged to be appropriate candidates for stimulant therapy based on rigorously diagnosed ADHD using DSM-IV criteria, were randomized into two open-label treatment groups (methylphenidate or mixed amphetamine salts). Each subject provided a blood sample before initiation of treatment and after 3 months of treatment. Pretreatment and posttreatment frequencies of chromosomal aberrations, micronuclei, and sister chromatid exchanges were determined for each subject.Results: Sixty-three subjects enrolled in the study; 47 subjects completed the full 3 months of treatment, 25 in the methylphenidate group and 22 in the amphetamine group. No significant treatment-related increases were observed in any of the three measures of cytogenetic damage in the 47 subjects who completed treatment or the 16 subjects who did not.Conclusions: Earlier findings of methylphenidate-induced chromosomal changes in children were not replicated in this study. These results add to the accumulating evidence that therapeutic levels of methylphenidate do not induce cytogenetic damage in humans. Furthermore, our results indicate that amphetamine-based products do not pose a risk for cytogenetic damage in children. [ABSTRACT FROM AUTHOR]

The National Toxicology Program (NTP) Center for the Evaluation of Risks to Human Reproduction (CERHR) conducted an evaluation of the potential for bisphenol A to cause adverse effects on reproduction and development in humans. The CERHR Expert Panel on Bisphenol A completed its evaluation in August 2007. CERHR selected bisphenol A for evaluation because of the: widespread human exposure; public concern for possible health effects from human exposures; high production volume; evidence of reproductive and developmental toxicity in laboratory animal studies Bisphenol A (CAS RN: 80-05-7) is a high production volume chemical used primarily in the production of polycarbonate plastics and epoxy resins. Polycarbonate plastics are used in some food and drink containers; the resins are used as lacquers to coat metal products such as food cans, bottle tops, and water supply pipes. To a lesser extent bisphenol A is used in the production of polyester resins, polysulfone resins, polyacrylate resins, and flame retardants. In addition, bisphenol A is used in the processing of polyvinyl chloride plastic and in the recycling of thermal paper. Some polymers used in dental sealants and tooth coatings contain bisphenol A. The primary source of exposure to bisphenol A for most people is assumed to occur through the diet. While air, dust, and water (including skin contact during bathing and swimming) are other possible sources of exposure, bisphenol A in food and beverages accounts for the majority of daily human exposure. The highest estimated daily intakes of bisphenol A in the general population occur in infants and children. The results of this bisphenol A evaluation are published in an NTP-CERHR Monograph that includes the (1) NTP Brief and (2) Expert Panel Report on the Reproductive and Developmental Toxicity of Bisphenol A. Additional information related to the evaluation process, including the peer review report for the NTP Brief and public comments received on the draft NTP Brief and the final expert panel report, are available on the CERHR website (http://cerhr.niehs.nih.gov/). See bisphenol A under "CERHR Chemicals" on the homepage or go directly to http://cerhr.niehs. nih.gov/chemicals/bisphenol/bisphenol.html). The NTP reached the following conclusions on the possible effects of exposure to bisphenol A on human development and reproduction. Note that the possible levels of concern, from lowest to highest, are negligible concern, minimal concern, some concern, concern, and serious concern. The NTP has some concern for effects on the brain, behavior, and prostate gland in fetuses, infants, and children at current human exposures to bisphenol A. The NTP has minimal concern for effects on the mammary gland and an earlier age for puberty for females in fetuses, infants, and children at current human exposures to bisphenol A. The NTP has negligible concern that exposure of pregnant women to bisphenol A will result in fetal or neonatal mortality, birth defects, or reduced birth weight and growth in their offspring. The NTP has negligible concern that exposure to bisphenol A will cause reproductive effects in non-occupationally exposed adults and minimal concern for workers exposed to higher levels in occupational settings. NTP will transmit the NTP-CERHR Monograph on Bisphenol A to federal and state agencies, interested parties, and the public and make it available in electronic PDF format on the CERHR web site (http://cerhr.niehs.nih.gov) and in printed text or CD from CERHR.

The National Toxicology Program (NTP) Center for the Evaluation of Risks to Human Reproduction (CERHR) conducted an updated evaluation of the potential for DEHP to cause adverse effects on reproduction and development in humans. The first CERHR expert panel evaluation of DEHP was completed in 2000 by the Phthalates Expert Panel. CERHR selected DEHP for an updated evaluation because of: (1) widespread human exposure, (2) public and government interest in adverse health effects, (3) recently available human exposure studies, and (4) the large number of relevant toxicity papers published since the earlier evaluation. DEHP (CAS RN: 117-81-7) is a high production volume chemical used as a plasticizer of polyvinyl chloride in the manufacture of a wide variety of consumer goods, such as building products, car products, clothing, food packaging, children's products (but not in toys intended for mouthing), and in medical devices made of polyvinyl chloride. The public can be exposed to DEHP by ingesting food, drink or dust that has been in contact with DEHP-containing materials, by inhaling contaminated air or dust, or by undergoing a medical procedure that uses polyvinyl chloride medical tubing or storage bags. It is estimated that the general population of the United States is exposed to DEHP levels ranging from 1 to 30 microg/kg bw/day (micrograms per kilogram body weight per day). The results of this DEHP update evaluation are published in an NTP-CERHR monograph that includes: (1) the NTP Brief, (2) the Expert Panel Update on the Reproductive and Developmental Toxicity of DEHP, and (3) public comments on the expert panel report. The NTP reached the following conclusions on the possible effects of exposure to DEHP on human development and reproduction. Note that the possible levels of concern, from lowest to highest, are negligible concern, minimal concern, some concern, concern, and serious concern. There is serious concern that certain intensive medical treatments of male infants may result in DEHP exposure levels that adversely affect development of the male reproductive tract. DEHP exposure from medical procedures in infants was estimated to be as high as 6000 microg/kg bw/day. There is concern for adverse effects on development of the reproductive tract in male offspring of pregnant and breast feeding women undergoing certain medical procedures that may result in exposure to high levels of DEHP. There is concern for effects of DEHP exposure on development of the male reproductive tract for infants less than one year old. Diet, mouthing of DEHP-containing objects, and certain medical treatments may lead to DEHP exposures that are higher than those experienced by the general population. There is some concern for effects of DEHP exposure on development of the reproductive tract of male children older than one year. As in infants, exposures of children to DEHP may be higher than in the general population. There is some concern for adverse effects of DEHP exposure on development of the male reproductive tract in male offspring of pregnant women not medically exposed to DEHP. Although DEHP exposures are assumed to be the same as for the general population, the developing male reproductive tract is sensitive to the adverse effects of DEHP. There is minimal concern for reproductive toxicity in adults exposed to DEHP at 1 - 30 microg/kg bw/day. This level of concern is not altered for adults medically exposed to DEHP. NTP will transmit the NTP-CERHR Monograph on DEHP to federal and state agencies, interested parties, and the public and it will be available in electronic PDF format on the CERHR web site http://cerhr.niehs.nih.gov and in printed text or CD-ROM from the CERHR.

An increase in the germ line mutation rate in humans will result in an increase in the incidence of genetically determined diseases in subsequent generations. Thus, it is important to identify those agents that are mutagenic in mammalian germ cells. Acrylamide is water soluble, absorbed and distributed in the body, chemically reactive with nucleophilic sites, and there are known sources of human exposure. Here we review all seven published studies that assessed the effectiveness of acrylamide or its active metabolite, glycidamide, in inducing transmitted reciprocal translocations or gene mutations in the mouse. Major conclusions were (a) acrylamide is mutagenic in spermatozoa and spermatid stages of the male germ line; (b) in these spermatogenic stages acrylamide is mainly or exclusively a clastogen; (c) per unit dose, i.p. exposure is more effective than dermal exposure; and (d) per unit dose, glycidamide is more effective than acrylamide. Since stem cell spermatogonia persist and may accumulate mutations throughout the reproductive life of males, assessment of induced mutations in this germ cell stage is critical for the assessment of genetic risk associated with exposure to a mutagen. The two specific-locus mutation experiments which studied the stem cell spermatogonial stage yielded conflicting results. This discrepancy should be resolved. Finally, it is noted that no experiments have studied the mutagenic potential of acrylamide to increase the frequency of transmitted mutational events following exposure in the female germ line.

The National Toxicology Program (NTP) Center for the Evaluation of Risks to Human Reproduction (NTP-CERHR) was established by the NTP and the National Institute of Environmental Health Sciences (NIEHS) in 1998 to address the impact of chemical exposures on human reproductive and developmental health and to serve as an environmental and reproductive health resource for government agencies and the general public. The purpose of this report is to provide an overview of the Center activities and a summary of NTP conclusions on chemicals evaluated during this time period. CERHR evaluations involve the critical review of reproductive, developmental, and other relevant toxicity data by independent panels of scientists. The products of these evaluations are expert panel reports. The public has opportunities to provide oral comments at the panel meeting and written comments on draft and final expert panel reports. The NTP evaluates these comments, the conclusions of the expert panel, and any new data not available at the time of the panel meeting, and prepares an NTP brief that describes in plain language the NTP's conclusions on the reproductive and developmental hazard from specified chemical exposures. The NTP brief, expert panel report, and public comments comprise the NTP monograph on the chemical. Monographs are sent to federal regulatory agencies, the NTP Executive Committee, and the NTP Board of Scientific Counselors, and are publicly available. Over the last five years, CERHR conducted expert panel evaluations on 14 chemicals. At this time, 13 panel reports have been published and 12 NTP-CERHR monographs have been issued. Additionally, CERHR conducted a 2-day workshop on the role of thyroid hormones in reproductive and developmental health.

Acrylamide is an animal carcinogen and probable human carcinogen present in appreciable amounts in heated carbohydrate-rich foodstuffs. It is also a germ cell mutagen, inducing dominant lethal mutations and heritable chromosomal translocations in postmeiotic sperm of treated mice. Acrylamide's affinity for male germ cells has sometimes been overlooked in assessing its toxicity and defining human health risks. Previous investigations of acrylamide's germ cell activity in mice showed stronger effects after repeated administration of low doses compared with a single high dose, suggesting the possible involvement of a stable metabolite. A key oxidative metabolite of acrylamide is the epoxide glycidamide, generated by cytochrome P4502E1 (CYP2E1). To explore the role of CYP2E1 metabolism in the germ cell mutagenicity of acrylamide, CYP2E1-null and wild-type male mice were treated by intraperitoneal injection with 0, 12.5, 25, or 50 mg acrylamide (5 ml saline)(-1) kg(-1) day(-1) for 5 consecutive days. At defined times after exposure, males were mated to untreated B6C3F1 females. Females were killed in late gestation and uterine contents were examined. Dose-related increases in resorption moles (chromosomally aberrant embryos) and decreases in the numbers of pregnant females and the proportion of living fetuses were seen in females mated to acrylamide-treated wild-type mice. No changes in any fertility parameters were seen in females mated to acrylamide-treated CYP2E1-null mice. Our results constitute the first unequivocal demonstration that acrylamide-induced germ cell mutations in male mice require CYP2E1-mediated epoxidation of acrylamide. Thus, CYP2E1 polymorphisms in human populations, resulting in variable enzyme metabolic activities, may produce differential susceptibilities to acrylamide toxicities.

The objective of this research was to determine whether Novantrone, Oncovin, Velban, and Prednisone (NOVP) combination chemotherapy for Hodgkin's disease increases the frequencies of the specific types of aneuploid sperm that might elevate the risk of fathering a child with one of the major clinical aneuploidy syndromes, i.e., Down (disomy 21 sperm), Edward (disomy 18 sperm), Turner (nullisomy sex sperm), XYY (disomy Y sperm), triple X (disomy X sperm), or Klinefelter (XY sperm). A four-chromosome multicolor sperm fluorescence in-situ hybridization assay that simultaneously evaluates chromosomes 18, 21, X, and Y was applied to semen provided by four healthy men and to repeated samples of eight Hodgkin's disease patients before treatment, 35-50 days after treatment to examine the effects of treatment on male meiotic cells, and 1-2 years after treatment to measure the persistence of damage. There were chromosome-specific variations in baseline frequencies and significant inductions of all of the detectable types of sperm aneuploidies: XY sperm (14-fold increase), disomy 18 (7-fold), nullisomy sex (3-fold), disomy 21 (3-fold), and disomy X and Y (approximately 2-fold each). Disomy 21 was about twice as frequent as disomy 18, and neither showed a preferential segregation with a sex chromosome. Extrapolating across the genome, approximately 18% of sperm carried a numerical abnormality after NOVP treatment of meiotic cells. Induced effects did not persist to 1-2 years after treatment, suggesting that persistent spermatogonial stem cells were not sensitive to NOVP. These findings establish the hypothesis that conception shortly after certain chemotherapies can transiently increase the risks of fathering aneuploid pregnancies that terminate during development or result in the birth of children with major human aneuploidy syndromes.

The purpose of the study was to determine: 1) if male germ cells of Big Blue mice carrying newly induced mutations in the lacI transgene were effective in fertilization; 2) if offspring arising from such mutant sperm had the mutation in germ cells and multiple somatic tissues; and 3) how the frequency of mutants induced in the lacI transgene compared to the frequency induced in endogenous genes traditionally employed to study germ cell mutagenesis in mice. Male B6C3F(1) mice hemizygous for the lambda/lacI transgene were treated weekly with 100 mg/kg body weight of the mammalian germ cell mutagen N-ethyl-N-nitrosourea (ENU). The cumulative dose for each treated animal was 300 mg ENU/kg body weight. Ten weeks later the treated mice were mated to T stock females and the resulting offspring were screened for specific-locus mutations at six loci affecting external appearance, as well as for mutations in the lacI transgene in multiple somatic tissues and germ cells. Five offspring carrying recessive specific-locus mutations were observed among 597 offspring screened (mutant frequency = 139.6 x 10(-5) per locus). Four offspring carrying lacI mutations were observed among 280 offspring screened (mutant frequency = 35.7 x 10(-5) per locus (assuming 40 target loci)). Each of the four lacI mutant offspring carried a different mutation. Three of the mutations were A:T-->G:C transitions and one a G:C-->A:T transition. Consistent with the expectation that a mutation induced in a parental germ cell and transmitted to a conceptus would exist in every cell of the offspring, each mutant mouse had identical mutations in all somatic tissues sampled, as well as in its germ cells. These data provide preliminary evidence for the biological validity of assessing induced, heritable mutations using transgenic mice, without the need for generating an F(1) generation., (Copyright 2002 Wiley-Liss, Inc.)

Many human immunodeficiency virus (HIV)-infected persons receive prolonged treatment with DNA-reactive antiretroviral drugs. A prospective study was conducted of 26 HIV-infected men who provided samples before treatment and at multiple times after beginning treatment, to investigate effects of antiretrovirals on lymphocyte and sperm chromosomes and semen quality. Several antiretroviral regimens, all including a nucleoside component, were used. Lymphocyte metaphase analysis and sperm fluorescence in situ hybridization were used for cytogenetic studies. Semen analyses included conventional parameters (volume, concentration, viability, motility, and morphology). No significant effects on cytogenetic parameters, semen volume, or sperm concentration were detected. However, there were significant improvements in sperm motility for men with study entry CD4 cell counts >200 cells/mm(3), sperm morphology for men with entry CD4 cell counts < or =200 cells/mm(3), and the percentage of viable sperm in both groups. These findings suggest that nucleoside-containing antiretrovirals administered via recommended protocols do not induce chromosomal changes in lymphocytes or sperm but may produce improvements in semen quality.

Dominant-lethal tests [P.D. Sudman, J.C. Rutledge, J.B. Bishop, W.M. Generoso, Bleomycin: female-specific dominant lethal effects in mice, Mutat. Res. 296 (1992) 205-217] had suggested that Bleomycin sulfate (Blenoxane), BLM, might be a female-specific mutagen. While confirming that BLM is indeed a powerful inducer of dominant-lethal mutations in females that fails to induce such mutations in postspermatogonial stages of males, we have shown in a specific-locus test that BLM is, in fact, mutagenic in males. This mutagenicity, however, is restricted to spermatogonia (stem-cell and differentiating stages), for which the specific-locus mutation rate differed significantly (P<0.008) from the historical control rate. In treated groups, dominant mutations, also, originated only in spermatogonia. With regard to mutation frequencies, this germ-cell-stage pattern is different from that for radiation and for any other chemical studied to date, except ethylnitrosourea (ENU). However, the nature of the spermatogonial specific-locus mutations differentiates BLM from ENU as well, because BLM induced primarily (or, perhaps, exclusively) multilocus deletions. Heretofore, no chemical that induced specific-locus mutations in spermatogonia did not also induce specific-locus as well as dominant-lethal mutations in postspermatogonial stages, making the dominant lethal test, up till now, predictive of male mutagenicity in general. The BLM results now demonstrate that there are chemicals that can induce specific-locus mutations in spermatogonia without testing positive in postspermatogonial stages. Thus, BLM, while not female-specific, is unique, (a) in its germ-cell-stage specificity in males, and (b) in inducing a type of mutation (deletions) that is atypical for the responding germ-cell stages (spermatogonia).

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., (Copyright 2000 Wiley-Liss, Inc.)

The induction of micronucleated erythrocytes by diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) was investigated as part of a U.S. National Toxicology Program (NTP) evaluation of the subchronic toxicity of these chemicals. Analysis of peripheral blood smears from male and female B6C3F1 mice exposed to 17.5-140.0 mg DIC/kg/day by skin painting for 13 weeks revealed dose-related increases in the frequency of micronucleated normochromatic erythrocytes (MN-NCE) in both sexes. Results of a similar 13-week peripheral blood micronucleus (MN) test with DCC (1.5-12.0 mg/kg/day) were also positive, although the increases in MN-NCE were not as great as those observed with DIC. In contrast to the positive results of the subchronic skin-painting studies in mice, acute bone marrow MN studies with DIC and DCC in male F344 rats, using intraperitoneal (i.p.) injection, yielded negative results. Both the acute and the subchronic exposures included doses that produced clinical signs of toxicity. Acute mouse bone marrow MN tests with DIC administered in single or triple i.p. injection protocols were subsequently conducted to determine if the differing responses between mice and rats were due to species or protocol differences. The results of these acute tests were negative or equivocal. Because the subchronic studies produced positive results, it was hypothesized that these carbodiimides required multiple treatments over an extended period of time to produce an increase in MN-erythrocytes. To confirm the original response, a second dermal subchronic study was conducted with DIC; the protocol was modified to include sequential blood samplings to permit monitoring MN frequencies over time. The data demonstrated a small but consistent induction of micronucleated erythrocytes in mice treated with DIC by skin painting.

Water samples from several ponds in Minnesota were evaluated for their capacity to induce malformations in embryos of Xenopus laevis. The FETAX assay was used to assess the occurrence of malformations following a 96-hr period of exposure to water samples. These studies were conducted following reports of high incidences of malformation in natural populations of frogs in Minnesota wetlands. The purpose of these studies was to determine if a biologically active agent(s) was present in the waters and could be detected using the FETAX assay. Water samples from ponds with high incidences of frog malformations (affected sites), along with water samples from ponds with unaffected frog populations (reference sites), were studied. Initial experiments clearly showed that water from affected sites induced mortality and malformation in Xenopus embryos, while water from reference sites had little or no effect. Induction of malformation was dose dependent and highly reproducible, both with stored samples and with samples taken at different times throughout the summer. The biological activity of the samples was reduced or eliminated when samples were passed through activated carbon. Limited evidence from these samples indicates that the causal factor(s) is not an infectious organism nor are ion concentrations or metals responsible for the effects observed. Results do indicate that the water matrix has a significant effect on the severity of toxicity. Based on the FETAX results and the occurrence of frog malformations observed in the field, these studies suggest that water in the affected sites contains one or more unknown agents that induce developmental abnormalities in Xenopus. These same factors may contribute to the increased incidence of malformation in native species.

The cancer chemotherapy agent, and topoisomerase-II inhibitor, etoposide (VP-16) produced both recessive mutations at specific loci and dominants at other loci with peak frequencies in primary spermatocytes, a cell type in which the topo-II gene has been shown to be activated. Etoposide thus differs from all other chemicals whose germ-cell-stage specificity has been analyzed. No effects of etoposide exposure of spermatogonial stem cells ( approximately 15, 000 offspring scored) were detectable by either mutagenicity or productivity endpoints. The significant mutagenic response that followed exposure of poststem-cell stages ( approximately 25,000 offspring scored) showed a clear peak, with three of four specific-locus mutants, and three of four dominant mutants conceived during weeks 4 or 5 (days 22-35) post-injection, a period that also encompassed the dominant-lethal peak. For this period, the induced specific-locus rate (with 95% confidence limits) at a weighted-average exposure of 75.1 mg etop/kg was 59.5 (14.6, 170. 9)x10-6/locus. At least 3 of the 4 specific-locus mutations were deletions, paralleling findings with etoposide or analogs in other test systems where a recombinational origin of the deletions has been suggested. Because, unlike other chemicals that induce deletions in male germ cells, etoposide is effective in stages normally associated with recombinational events, it will be of interest to determine whether this chemical can affect meiotic recombination., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Tamoxifen (TAM), an antiestrogen, is widely used to treat hormone-dependent breast cancer in post-menopausal women. TAM may be used as a chemopreventive agent in women of child-bearing age; however, few data exist describing potential TAM-induced fetal toxicity. In support of the National Toxicology Program's characterization of reproductive and developmental effects of TAM, this work describes an analytical technique utilizing capillary electrophoresis (CE) for the detection of circulating levels of TAM, N-desmethyltamoxifen (DMT), and 4-hydroxytamoxifen (4-HT) in maternal rodent serum. Greater than 90% of 3H-labeled TAM was extractable from serum using 98:2 hexane-isoamyl alcohol. Optimum separation of TAM, DMT, and 4-HT was obtained on a 57 cmx50 microm capillary using a nonaqueous buffer system of 1:1 methanol-acetonitrile containing 50 mM ammonium acetate and 1% acetic acid. 4-Dimethylaminopyridine was used as internal standard. Temperature and voltage were optimized at 40 degrees C and 15 kV, respectively. The limit of detection of TAM by UV detection at 214 nm was approximately 800 amol. TAM and DMT were confirmed in serum of female rats 4 h following a single oral dose of 120 mg/kg. Transplacental exposure of TAM to fetal tissue will be evaluated using this technique.

Three confirmed mouse germ cell mutagens, ethyl nitrosourea (ENU), isopropyl methanesulphonate (iPMS) and methyl methanesulphonate (MMS), have been evaluated for their activity as mutagens to the germ cell DNA of two strains of transgenic mice (lac I, Big Blue and LacZ, Muta Mouse). Both testicular DNA and epididymal sperm DNA were evaluated. A range of sampling times was studied, from 3 days post-dosing to 100 days post-dosing. ENU and iPMS were mutagenic to both testicular DNA and epididymal sperm DNA. Mutant frequencies were higher for both chemicals in DNA recovered from testicular tissue than in epididymal sperm DNA. Likewise, mutant frequencies were higher for both DNA samples at the later sampling times. MMS was not mutagenic under any condition of test. A good level of qualitative agreement in test results was seen for the two assays and for the same assays conducted in different laboratories. The level of quantitative agreement was not as high, but was, nonetheless, generally good. Recommendations for the future conduct of transgenic rodent germ cell mutation assays are made. The test data are discussed within the context of the larger question of how such assays should be integrated into the chemical hazard assessment process.

A collaborative study to systematically assess transgenic mouse mutation assays as screens for germ cell mutagens has been conducted. Three male mouse germ cell mutagens (ENU, iPMS and MMS) were selected for testing. This paper provides a brief review of the effects reported for those 3 chemicals in the most commonly used non-transgenic germ cell mutagenicity assays, namely the dominant lethal, heritable translocation, and specific locus tests. Additionally, information on the DNA reactivity and the molecular nature of mutations induced by these chemicals is summarized.

Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173-213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20- to 24-hour sampling time. While positive results were obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially "gaps"; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as "gaps" by the Japanese system were classified as "breaks" in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

Because of rampant concern that estrogenic chemicals in the environment may be adversely affecting the health of humans and wildlife, reliable methods for detecting and characterizing estrogenic chemicals are needed. It is important that general agreement be reached on which tests to use and that these tests then be applied to the testing of both man-made and naturally occurring chemicals. As a step toward developing a comprehensive approach to screening chemicals for estrogenic activity, three assays for detecting estrogenicity were conducted on 10 chemicals with known or suspected estrogenic activity. The assays were 1) competitive binding with the mouse uterine estrogen receptor, 2) transcriptional activation in HeLa cells transfected with plasmids containing an estrogen receptor and a response element, and 3) the uterotropic assay in mice. The chemicals studied were 17 beta-estradiol, diethylstilbestrol, tamoxifen, 4-hydroxytamoxifen, methoxychlor, the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), endosulfan, nonylphenol, o,p'-DDT, and kepone. These studies were conducted to assess the utility of this three-assay combination in the routine screening of chemicals, or combinations of chemicals, for estrogenic activity. Results were consistent among the three assays with respect to what is known about the estrogenic activities of the chemicals tested and their requirements for metabolic activation. By providing information on three levels of hormonal activity (receptor binding, transcriptional activation, and an in vivo effect in an estrogen-responsive tissue), an informative profile of estrogenic activity is obtained with a reasonable investment of resources.

Specific-locus studies have shown chlorambucil (CHL) and bleomycin (BLE) to be mutagenic in mouse oocytes, almost doubling the number of chemicals previously known to induce mutations in females. The overall CHL-induced mutation rate in oocytes is, however, one order of magnitude below that for male meiotic and postmeiotic stages, and only 1/50 that for early spermatids. For BLE, no specific-locus data for males are available for comparison, but the chemical had earlier been found negative for dominant-lethal induction in males. Both BLE and CHL were significantly mutagenic only in mature and maturing oocytes. In keeping with an earlier report, BLE produced a high incidence of dominant lethals in these stages. CHL failed to induce dominant lethals, indicating that for mature and maturing oocytes, in contrast with results for males, sensitivity to dominant-lethal mutations is not a prerequisite for induction of specific-locus mutations. Exposure of immature oocytes to either BLE or CHL produced neither dominant lethals nor significant induction of specific-locus mutations; however, CHL gave evidence of killing immature oocytes. By contrast, BLE, which has been considered a radiomimetic chemical, does not appear to kill immature oocytes and thus differs markedly from radiation exposures equivalent for dominant-lethal induction. Therefore, the failure to recover specific-locus mutations cannot be ascribed to cell selection resulting from oocyte killing, as has sometimes been done for radiation. Adding results on the nature of the CHL- and BLE-induced mutations to prior information, the estimated minimum proportion of large DNA lesions induced in oocytes by chemicals becomes 35.3%, significantly different from the corresponding figure (approximately 70%) for radiations. For chemical treatments, the oocyte proportion is highly significantly above the 3.6% induced in spermatogonia, but only on the borderline of statistically significant difference from that induced in postspermatogonial stages.

A flow chart is presented as a recommended sequence of tests to predict the carcinogenic hazard, and to predict and quantify the mutagenic hazard to germ cells of chemicals to humans. Ten associated principles of testing for these endpoints are also suggested. These recommendations are the result of a meeting convened under the auspices of the International Programme on Chemical Safety (IPCS), as part of their project on 'Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals'. The meeting was held at Carshalton, Surrey, from 13-17 February 1995.

Chemicals used in the treatment of cancer include several that are potent mutagens in a range of in vitro and in vivo assays. For some, genetic effects have also been demonstrated in humans, detected as chromosomal aberrations in peripheral lymphocytes. Because (1) many of these agents are confirmed mutagens, (2) humans are exposed to them in relatively high doses, and (3) an increasing number of early cancer victims are surviving to reproductive age, it is important that information be available on the genetic and reproductive hazards associated with exposure to these agents. Chlorambucil and melphalan are structurally related chemicals that are included in our efforts to identify and assess such hazards among cancer chemotherapy agents. To date, both have been reported to induce specific locus mutations in germ cells of male mice (Russell et al., 1989; Russel et al., 1992b) and melphalan is one of very few chemicals shown to induce such mutations in spermatogonial stem cells. More recently, both chemicals were found to have strong reproductive effects in female mice (Bishop and Generoso, 1995, in preparation). In the present studies, these chemicals were tested for the induction of dominant lethal mutations and heritable translocations in male mice. Both chemicals were found to have reproductive effects attributable to cytotoxicity in specific male germ cell stages and to induce dominant lethal mutations and heritable translocations in postmeiotic germ cells, particularly in mid to early stage spermatids. Thus, relatively extensive data are now available for assessing the genetic and reproductive hazards that may result from therapeutic exposures to these chemicals.

Phenolphthalein was tested for the induction of micronucleated erythrocytes in mice. Results of an initial investigation revealed significant, dose-related increases in micronucleated polychromatic erythrocytes (MN-PCE) and normochromatic erythrocytes (MN-NCE) in peripheral blood samples of male and female mice exposed to 0.6% to 5% phenolphthalein (approximately 1100 to 10,000 mg/kg/day) in feed for 90 days (Dietz et al., 1992). Results from a second long-term feed study with Swiss CD-1 mice confirmed this effect. However, administration of comparable doses of phenolphthalein by corn oil gavage on two consecutive days gave negative results in a mouse bone marrow micronucleus test. Subsequent tests were performed to clarify the conflicting results seen in the chronic exposure, dosed-feed, peripheral blood studies and the acute, corn oil gavage, bone marrow studies. Phenolphthalein was administered to male B6C3F1 mice in feed (3%) for 14 days. Peripheral blood samples taken at 4, 7, and 14 days all showed significant increases in micronucleated PCE; bone marrow samples taken on days 7 and 14 also were clearly positive for micronucleus induction. Therefore, comparable results were obtainable from both bone marrow and peripheral blood analyses. Because of the negative results in the two-exposure gavage test, additional tests were then designed to investigate the effects of bolus vs continuous dosing, feeding vs gavage administration, and corn oil vs feed as a carrier for phenolphthalein. Results of these tests indicated that the rate of exposure to phenolphthalein affects the frequency of induced MN-PCE and that micronucleated erythrocytes can be induced by phenolphthalein either by feeding or by corn oil gavage administration. In all the acute exposure studies, relatively high doses of phenolphthalein (2000-6000 mg/kg/day for at least 2 days) were required to induce micronuclei. The positive results obtained with phenolphthalein in vivo were consistent with the results of an in vitro chromosomal aberration test in Chinese hamster ovary cells, where dose-related increases in aberrations were noted only in cells treated in the presence of induced rat liver S9.

Design features that adjust and account for excess variation in a transgenic mouse mutation assay based on a lacI target transgene from E. coli are considered. These features include proper identification of plate, packaging reaction, and animal identifier codes throughout the experimental and analysis phases of the study, "blocking" of exposed and unexposed animals when preparing and plating multiple packaging reactions from the same genomic DNA sample, separating sectored mutant plaques and complete mutant plaques before performing any quantitative analyses, and testing for sources of excess variation attributable to features of the experimental protocol--such as plate-to-plate (within packaging reactions), packaging reaction-to-packaging reaction (within animals), and animal-to-animal (within study). Control and ethylnitrosourea-treated animal data are presented from a fully designed study in the lacI assay. The study design incorporates many of these experimental principles. Statistical methods to identify excess variability are noted, and the designed study data are used to illustrate the types of variability encountered in practice. A standard statistical test for two-sample testing is highlighted, from which recommendations are made for sample size selection in future studies.

Tests for the induction of chromosomal aberrations (ABS) and micronuclei (MN) in bone marrow cells of mice have been conducted on 65 chemicals. Although these tests were not conducted with the purpose of comparing the outcomes of these two in vivo genetic toxicity endpoints, the availability of these test results permits such a comparison. Based on studies to date, results from the 2 tests agree for more than 80% of the chemicals; 17 gave positive results in both tests, and 36 gave negative results in both. Seven chemicals were positive only for ABS and 5 were positive only for MN. Three chemicals that were originally concluded to be positive for ABS but not for MN were found to induce MN when the MN protocol was modified to more closely reflect the ABS protocol. Among the 12 chemicals for which there are discrepant results, there are only 2 for which the difference is convincing. One of these, selenium sulfide (MN negative, ABS positive) remains an enigma; further studies are being conducted. The second, isoprene (MN positive, ABS negative) will be difficult to pursue because the studies reported here were done by inhalation exposure. Based on the outcomes of these comparisons, protocol factors, rather than endpoint specificity, appear to be the major source of discrepant test results. Thus, these results do not support a recommendation that both tests be conducted in a primary testing scheme for genetic toxicity.

This special issue attempts to provide a fresh perspective on the importance of germ-cell mutagenesis studies and restate the questions and challenges inherent in efforts to minimize the incidence of human genetic diseases. We are working in a time when rapidly advancing molecular technologies provide the tools that permit a more detailed understanding of germ-cell mutagenesis and genetic disease. Meanwhile, discoveries of new genetic disease phenomena challenge our abilities to conceive and develop research models for their study. It is hoped that the collection of articles in this issue will serve to stimulate interest in scientists of varied disciplines and help focus those interests on the issues surrounding the relationship between environmental mutagens and human genetic disease.

The two tests considered by the Working Group were the mammalian germ cell cytogenetic assay and the rodent dominant lethal test. It was agreed that both tests were mainly used for identification of germ cell hazards, however, that the commonly applied protocol of the dominant lethal assay often supplied information for hazard characterization such as sensitivity of particular developmental stages of male germ cells. No particular species or strains were indicated. Concurrent solvent controls were regarded as indispensable for both tests. In the discussion of the mammalian germ cell cytogenetic assay, harmonization was obtained to a large extent with the cytogenetic bone marrow assay regarding the number of animals (5), the number of cells analyzed per animal (200), the highest exposure dose (MTD) and sampling times (twice within 24 and 48 h after dosing). However, it was pointed out that only the single acute exposure was adequate for the mammalian germ cell cytogenetic assay. Furthermore, it was stated that only structural chromosome aberrations could be analyzed and that it was not informative to score polyploidies or aneuploidies. In the discussion of the rodent dominant lethal test, it was stated that the assay was generally performed with treated males, however, increasing concern about female specific effects required that a protocol for female dominant lethal testing should be developed and validated. Acute and subacute treatment schedules were considered equally acceptable. It was regarded as highly important that the entire male germ cell development from meiosis to mature sperm was covered in the test protocol either by the appropriate mating schedules after single dosing or by subchronic dosing during the respective period. Postimplantation loss, preimplantation loss and fertility rate were the main parameters to be assessed in the rodent dominant lethal tests. It was agreed that the size of the experiment depended on the spontaneous frequency of dead implants, the mating scheme and the statistical design of the experiment.

Human genetic disorders constitute a major public health burden in this country and around the world. The possibility that exposures to mutagenic environmental agents may result in induced genetic damage in human germ cells and thereby increase the incidence of genetic disease has been investigated in research laboratories and in epidemiology studies for decades. The capacity of ionizing radiation and some chemicals to induce transmissible genetic damage in the germ cells of laboratory mammals has been clearly demonstrated and extensively investigated. To date, no clear evidence of such effects in humans has been reported although increased frequencies of chromosomal aberrations have been detected in human sperm following exposures to radiation or chemotherapeutic agents. Evolving methods to detect molecular changes in DNA offer to improve our abilities to detect induced genetic changes. The integration of these methods into mutation epidemiology studies promises to help resolve some of the questions regarding human genetic risk.

Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined with the goal of identifying sources of excess variation in the observed mutant fractions. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability. Data from two laboratories are evaluated, using various statistical methods to identify excess variability. Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues. Further study is encouraged to examine the validity and implications of this time/storage-related effect.

Because genetically based diseases have a major impact on human health, the National Institute of Environmental Health Sciences (NIEHS) has conducted a research and testing program for more than a decade to address chemical induction of heritable genetic damage in the germ cells of mammals. Although most genetic disease results from preexisting mutations, a portion is due to the occurrence of new mutations. The supposition that exposure to mutagenic chemicals contributes to the occurrence of new mutations in the human population is strongly supported by the results from animal models. Such studies clearly demonstrate the potential of environmental chemicals to induce mutations in both somatic and reproductive cells of mammals. This NIEHS program has become a leader in the identification of genetic hazards in the environment and in the acquisition of animal model data used by regulatory agencies in assessing genetic risks to human health.

F- is not mutagenic in standard bacterial systems, but produces chromosome aberrations and gene mutations in cultured mammalian cells. Although there is disagreement in the literature concerning the ability of F- to induce chromosome aberrations in cultured human and rodent cells, the weight of the evidence leads to the conclusion that F- exposure results in increased chromosome aberrations in these test systems. NaF induced primarily chromatid gaps and chromatid breaks, indicating that the rodent cells are responsive in the G2 stage of the cell cycle. In contrast, studies with synchronized human cells indicated that the S phase was the most sensitive. If F- does have a cell cycle-specific effect, it could be expected that differences in the cell treatment and harvest protocols could lead to conflicting results for the induction of chromosome aberrations. Gene mutations were produced in cultured rodent and human cells in the majority of the studies. Unfortunately, a number of the in vitro and in vivo cytogenetic studies are of questionable utility because of the protocols used, the quality of the responses reported, or the interpretations of the data. The conflicting results in the in vivo cytogenetic studies are difficult to reconcile. There are reports of increased chromosome aberrations in rat bone marrow and testes, but other studies, using similar protocols and dose ranges, have reported no induced chromosome damage. Although some of the studies were performed at toxic levels of F-, other studies, including those that showed positive results, were at F- concentrations (1-5 ppm) equivalent to human exposure levels. In the majority of studies that were reported to be positive, there were high background frequencies, or the investigators reported categories of nuclear or chromosome damage that are difficult to interpret. Interestingly, many of the positive results were obtained when anaphase cells were scored, whereas similar treatment protocols in other laboratories yielded negative results when metaphase cells were the only cell type examined. It is difficult, without additional data, to determine the reasons for finding chromosome breaks in anaphase, but not metaphase, cells. Other reports have presented insufficient information to allow adequate evaluations. Therefore, at this time, the question of whether F- produces chromosome damage in vivo should be considered unresolved.

Forty-nine chemicals were tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure. Twenty-five rodent carcinogens and 24 noncarcinogens were selected randomly from the 44 carcinogens and 29 noncarcinogens used by Tennant et al. (Science 236:933-941, 1987) to evaluate the performance of four in vitro genetic toxicity tests. As in that study of in vitro tests, the micronucleus tests were conducted with coded chemicals and test results (positive or negative) were determined prior to decoding. This study was conducted as part of an effort to assess the ability of the micronucleus test to discriminate between rodent carcinogens and noncarcinogens and to determine its potential role, in combination with other short-term tests, in identifying genotoxic chemicals that present a carcinogenic hazard. Nine chemicals were judged to be positive in the micronucleus test. This relatively low number of positive results, along with published and unpublished results from rodent micronucleus and chromosome aberration assays on several of these 49 chemicals, contributed to the conclusion that a single micronucleus test protocol is not adequate to detect all chemicals capable of inducing chromosomal damage in the bone marrow. However, a combination of two relatively simple assays such as the Salmonella and micronucleus tests can provide important information on the genetic toxicity of test chemicals and may provide guidance on the need for and the nature and extent of future toxicity studies.