Male-mediated Developmental Toxicity

By Diana Anderson, Martin H Brinkworth

The Royal Society of Chemistry

Copyright © 2007 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-847-2

Contents

Foreword Marcello Lotti MD, v,
Introductory Scientific Presentations,
Chapter 1 Epigenetic Transgenerational Actions of Endocrine Disruptors through the Male Germ-Line Michael K. Skinner,
Chapter 2 Reproductive Outcomes among Men Treated for Cancer John J. Mulvihill and Timothy J. Garlow,
Chapter 3 Cancer in Siblings of Children with Cancer in the Nordic Countries: A Population-Based Cohort Study Paediatric Cancer: An Indicator of Familial Cancer Risk? Jeanette Falck Winther,
Chapter 4 What Harms the Developing Male Reproductive System? Michael Joffe,
Chapter 5 Links Between Paternal Smoking and Childhood Cancer Tom Sorahan,
Chapter 6 Feasibility Study of Metal Effects on the X:Y Ratio in Human Sperm Wendie A. Robbins, Karen E. Young, Fusheng Wei and The Boron Epidemiology Research Group,
Chapter 7 Use of the Sperm Chromatin Structure Assay (SCSAs) as a Diagnostic Tool in the Human Infertility Clinic Donald P. Evenson and Regina L. Wixon,
Chapter 8 Safety of Sperm for Use in ICSI D. Sakkas, E. Seli, D. Bizzaro, G.C. Manicardi, A. Jakab and G. Huszar,
Chapter 9 Male-Mediated F1 Effects in Mice Exposed to Di(2-ethylhexyl)phthalate (DEHP) Mazgorzata M. Dobrzynska, Urszula Czajka and Ewa J. Tyrkiel,
Chapter 10 Prevention of Adverse Effects of Cancer Treatment on the Germline Marvin L. Meistrich, Zhen Zhang, Karen L. Porter, Olga U. Bolden-Tiller and Gunapala Shetty,
Chapter 11 Molecular Changes in Sperm and Early Embryos after Paternal Exposure to a Chemotherapeutic Agent Bernard Robaire, Alexis M. Codrington and Barbara F. Hales,
Chapter 12 Transmissible Genetic Risk Causing Tumours in Mice and Humans Taisei Nomura,
Chapter 13 Heritable Effects on DNA Damage Following Paternal F0 Germline Irradiation Ming-Wen Li and Janet E. Baulch,
Chapter 14 Influence of DNA Methylation and Genomic Imprinting in the Male Germ Line on Pregnancy Outcome Jacquetta M. Trasler,
Chapter 15 Information Content of Ejaculate Spermatozoa and its Potential Utility in Toxicology and Infertility Based Research Programmes David Miller, Martin Brinkworth and David Iles,
Chapter 16 Origin of Paternal Mutations James F. Crow,
Chapter 17 Redox Regulation of DNA Damage in the Male Germ Line R.J. Aitken, S.D. Roman, M.A. Baker and G. De Iuliis,
Chapter 18 Advances in the Direct Measurements of Partial Chromosomal Duplication, Deletions and Breaks in Human and Murine Sperm by Sperm FISH Andrew J. Wyrobek, Thomas E. Schmid, Jack Bishop and Francesco Marchetti,
Chapter 19 Radiation-induced Transgenerational Instability in Mice Yuri E. Dubrova,
Chapter 20 New Genetic Information Generated by Endogenous Reverse Transcription in Sperm Cells Corrado Spadafora,
Chapter 21 Sperm Abnormalities in Exposed Humans Radim J. Sram and Jiri Rubes,
Chapter 22 Oestrogenic Compounds and Oxidative Stress Diana Anderson, Eduardo Cemeli, Thomas E. Schmid, Adolf Baumgartner, Martin H. Brinkworth and John M. Wood,
Chapter 23 DNA Repair Capacities in Testicular Cells of Rodents and Man Gunnar Brunborg, Nur Duale, Julie Tesdal Haaland, Christine Bjørge, Erik Søderlund, Erik Dybing, Richard Wiger and Ann-Karin Olsen,
Chapter 24 3rd International Congress on Male-Mediated Development Toxicity: Closing Panel Discussion Jack Bishop and Barbara F. Hales,

CHAPTER 1

Epigenetic Transgenerational Actions of Endocrine Disruptors through the Male Germ-Line

MICHAEL K. SKINNER

Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman WA 99164-4231

1.1 Review

Embryonic exposure to environmental factors has been shown to cause adult onset disease, but few have looked at the second F2 generation. Examples include the late embryonic and early postnatal exposure to cyclophosphamide causing embryonic defects, embryonic nutritional defects causing immune defects, diethylstilbesterol (DES) causing female reproductive tract abnormalities, and other endocrine disruptors causing male reproductive defects. Studies suggest effects of environmental factors on the first generation. Any transgenerational phenotype would require transmission through the germ-line.

A recent observation demonstrated that the exposure of a pregnant rat transiently to endocrine disruptors caused a spermatogenic cell defect and sub-fertility in the F1 generation and all subsequent generations examined (F1–F4).The endocrine disruptors used were the anti-androgenic fungicide vinclozolin used in the fruit (e.g., wine) industry and the pesticide methoxychlor used to replace DDT. The critical exposure period was at the time of sex determination and the transgenerational phenotype was transmitted through the male germ-line. The phenotype of increased spermatogenic cell apoptosis, decreased sperm numbers and sperm motility was observed in greater than 90% of all males of all the generations examined. When the animals were allowed to age up to 1 year additional diseases developed including cancer, prostate disease, kidney disease, and immune cell defects. A high frequency of transmission was observed in all generations examined for all the disease states.

The frequency of the transgenerational phenotype was such that a DNA sequence mutational event could not be involved. The random nature of a DNA sequence mutation has a phenotype typically less than 1 % and this often declines in subsequent generations. An epigenetic mechanism is found to be involved due to the frequency of the phenotype. To support these conclusion, two genes were identified in the sperm that had altered methylation patterns associated with the transgenerational phenotype discussed. Therefore, the endocrine disruptors appear to induce an epigenetic transgenerational disease condition for four generations through the male germ-line. The epigenetics appears to involve altered DNA methylation. Although most genes get re-set in early embryonic development, a subset of genes called imprinted genes maintains their DNA methylation pattern which appears to be permanently programmed. In contrast to all somatic cells the primordial germ cells undergo a de-methylation during migration and early colonization of the embryonic gonad, followed by a re-methylation starting at the time of sex determination in a sex-specific manner. The exposure of the pregnant mother at the time of sex determination appears to have altered the re-methylation of the germ-line and permanently re-programmed the imprinted pattern of DNA methylation. This provides a unique epigenetic mechanism to promote a transgenerational phenotype induced by an environmental factor.

Altered methylation of imprinted genes has been shown to promote disease states. Cancer and tumor development has also been shown to be involved in epigenetic alteration of DNA methylation. Therefore, the epigenetic reprogramming of the male germ-line causes numerous transgenerational disease states that can be explained by this epigenetic mechanism. The identification of the altered DNA methylation sites and associated genes will provide more insight into the proposed epigenetic transgenerational phenotype.

The level of endocrine disruptors used in the recent studies is higher than levels anticipated in the environment, such that conclusions regarding the toxicology of these endocrine disruptors are not possible. However, the important factor is the identification of this novel phenomenon, that an environmental factor can promote an epigenetic transgenerational phenotype. Due to this observation the potential hazards of environmental factors need to be carefully evaluated. If the exposure of your grandmother at mid-gestation to environmental toxins can cause a disease state in you with no exposure, and you will pass it on to your grandchildren, the potential hazards of environmental toxicants must be rigorously assessed. Transgenerational studies need to be performed in evaluating the toxicology of environmental compounds.

The epigenetic transgenerational phenotype also provides critical insights into disease etiology. Since a number of common disease states were induced, an epigenetic component of disease now needs to be seriously considered. In the event a major epigenetic component exists, the epigenetic background of an individual may be a major factor in susceptibility to disease development. Therefore, identification of the genes involved with altered methylation may provide essential new diagnostics to assess future onset of disease. This will allow new therapeutic targets and therapies to potentially prevent the onset of disease. This is a new paradigm in disease etiology that needs to be considered.

In a broader biological perspective, the ability of an environmental factor to cause a permanent genetic trait in all subsequent progeny of an affected individual can significantly impact our understanding of evolutionary biology. Currently, a DNA sequence mutation event that allows an adaptation and natural selection is considered the driving factor in evolutionary biology. However, the frequency of specific evolutionary events and regional influences on evolution suggests that an additional epigenetic mechanism should be considered. Although a DNA sequence mutational event will be important for evolutionary biology, an epigenetic component influenced by an environmental factor needs to be considered as an alternate factor that will help explain some aspects of evolutionary biology.

The epigenetic transgenerational actions of endocrine disruptors observed provide novel insights into several areas of biology. The ability of an environmental compound to promote a transgenerational phenotype suggests toxicology studies need to consider transgenerational elements of the actions of potential toxic agents. Future studies need to investigate the types of compounds that can induce the epigenetic effect. Currently we know anti-androgenic compounds can, but need to assess if other factors can as well. The toxicology studies need to be done to assess the minimum required dose to obtain a phenotype and compare this to potential environmental levels. This information will reveal if the levels in our environment are a problem. The epigenetic effects on the methylation state of specific genes needs to be determined to provide insights into the mechanisms of action of the environmental factors. In addition, these genes will provide potentially critical diagnostic markers and therapeutic targets for a variety of common diseases. The basic elements of disease etiology now need to consider epigenetic factors as markers and/or causal factors. In a broader context, the epigenetic transgenerational impact of environmental factors needs to be considered in the mechanisms involved in evolutionary biology. Epigenetics will likely be a much more important factor in biology than currently appreciated. Epigenetics is the next layer of complexity beyond the DNA sequence.

CHAPTER 2

Reproductive Outcomes among Men Treated for Cancer

JOHN J. MULVIHILL AND TIMOTHY J. GARLOW

Department of Pediatrics and General Clinical Research Center, University of Oklahoma, Oklahoma City OK 73104, USA

2.1 Why Study Cancer Survivors?

A report from the US Institute of Medicine and National Research Council showed that, as of the end of 2002, an estimated 10.1 million individuals in the US had received the diagnosis of a cancer (other than nonmelanotic skin cancer) in their lifetime; 40% of them were under age 65 years. One can assume half are male and hence estimate up to two million men in the US are cancer survivors with reproductive potential. Estimated another way, close to a half million men of reproductive age are added to US population each year, with the inclusion of recipients of organ transplants (large kidneys) who tend to receive some chemotherapy-like agents and the exclusion of men who die in within 5 years of diagnosis (Table 1).

The therapies for men with cancer comprise several categories: physical agents (namely, ionizing radiation), antimetabolites, alkylating agents, antibiotics, and alkaloids. These agents are potent mutagens since they are intended to interfere with DNA metabolism. They are often given not as a single agent, but rather as combinations. So, in contrast to the pure exposure of experimental investigations of male reproductive toxicity, the human exposure to cancer treatment is complex. But, unlike other human exposures of concern for mutagenicity, the timing and dosage of exposure to cancer therapy can be precisely known. Moreover, dosage can be independently corroborated by medical records and occasionally by direct in vivo measurements, for example, with blood levels of DNA adducts.

2.2 Endpoints

The endpoints that can be studied are those mentioned throughout this volume. Some are clinical endpoints that do not require laboratory collaboration such as spontaneous abortion, infertility, and genetic diseases, including birth defects and especially the so-called sentinel phenotypes. A sentinel phenotype is a clinical disorder or syndrome that occurs sporadically as a consequence of a single, highly penetrant mutant gene that is a dominant trait of some frequency and low fitness and that is uniformly expressed and accurately diagnosable, with minimal effort at or near birth. In Online Mendelian Inheritance in Man, there are about 40–80 traits that meet this definition. Together they have an estimated total frequency around one in a thousand. In short, sentinel phenotypes, such as achondroplasia or aniridia, are powerful endpoints, clinically relevant, almost certainly representing new mutation, and possibly attributed to environmental exposure. But, they are rare events and large numbers of exposed men would have to be counted to observe their offspring for such defects.

Other endpoints require laboratory collaboration: sperm analyses and, in offspring, chromosomal abnormalities and variations in proteins or nucleic acids. With many different gene tests on single subjects, fewer exposed people are needed to achieve statistical power. However, the relevance of molecular abnormalities to the health of clinically normal offspring is problematic.

2.3 Decreased Fertility

There are many barriers to reproduction by men (and women) after cancer and its treatment. The focus of this chapter is the narrow issue of testicular impairment and germ cell mutation. However, reproductive potential of cancer survivors could be limited by the many concomitant associations of the experience of cancer in childhood, adolescence, and early adulthood: impaired education, growth, and development, limited job opportunities and income, sterility per se, dismemberment or deformity due to therapy, or fear and uncertainty about the future.

Many variables influence even a single measure of reproduction or reproductive capacity, even one as discrete as gonadotrophin levels. For example, young men were studied who had received cyclophosphamide, not for cancer but for immunologic renal disease in childhood. In general, the frequency of gonadal dysfunction, as reflected by gonadotrophin levels in blood, increased with the dose of cyclophosphamide; but, it also varied with age at exposure or, more precisely, with the pubertal status. The post-pubertal testis is much more sensitive to sterilization by cyclophosphamide than the pre-pubertal testis, which is only slightly impaired at doses given for immune renal disease.

By contrast, there is a large male-female difference in reproductive outcomes among long-time survivors of Wilms’ tumor of the kidney. In a network of seven pediatric hospitals, 30% of 114 pregnancies of women who survived Wilms’ tumor and who underwent abdominal radiation had adverse outcomes, defined as fetal or neonatal death or birth weights under 2.5 kg. The rates of such outcomes among female survivors without abdominal radiation and among the wives of male survivors were 0% and 3%, respectively. A postal survey of British general practitioners who treated survivors of cancer diagnosed in childhood between 1946 and 1977, identified 20 Wilms’ tumor survivors. The birth weight of offspring of women who received abdominal radiation was 2584 g; among the wives of male survivors and women who did not undergo radiation, birthweight was 3146 g, a highly significant difference. In short, the offspring of men with Wilms’ tumor did not suffer these consequences.

2.4 The Five-Center Study

2.4.1 Design

To clarify several issues concerning late reproductive and other effects among cancer survivors, a team at the National Cancer Institute struck collaborations with five cancer registries around the US in the late 1970s. The first goal was to assemble a cohort of long-term survivors of childhood and adolescent cancer. Cases had either histologically confirmed cancer or clinically diagnosed brain tumors. Diagnosis took place from 1945 through 1974, under the age of 20 years. Finally, survival of 5 years and attainment of 21 years were required by an arbitrary date of study cutoff.

These criteria produced a special set of 2498 eligible cancer survivors. Because three decades of cancer treatment were included, half of the cases had surgery only; these represent a control group within the study itself that could be used to distinguish the effects of therapy from any determinants of cancer risk. One-third had radiotherapy only, and another third had some chemotherapy, often far less than the multiagent chemotherapy that pediatric oncologists presently give. Twenty-one percent of study subjects were pre-pubertal boys and girls, so we were able to do some comparisons between pre- and post-pubertal exposures. The collected data included an interview about interval medical and social history, infertility, any ill health of the offspring and, importantly, consent for records so that certain medical events, such as cancer, birth defects, and infertility, could be documented by actual medical records or death certificates. Cases’ permissions were also gained to interview their brothers and sisters, who were selected as controls in a ratio of two controls per one case.

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