🧬 PART EIGHT NOXIOUS AGENTS

796  NOXIOUS AGENTS BIOLOGY AND EPIDEMIOLOGY Cancer Treatment A majority of children and young adults who receive modern cancer treatment survive. Some treatments cause sterility, but in quite a number fertility is unscathed, or at any rate subsequently recovers (van Santen et al. 2020). For those who are potentially capable of having children, the question arises: Could there be an increased risk to have a child with a chromosomal abnormality? For most, in fact, the short answer may be apparently not. Longer answers follow. The chemotherapeutic agents and radiation used to rid the body of cancer are essentially cellular toxins, some of which specifically target DNA or the mitotic apparatus. Thus, the starting hypothesis is that the chromosomes in exposed bystander tissues (and thinking specifically of the gonad) could be vulnerable. The fact that these treatments can damage chromosomes is well known, and this is actually the basis of one of the in vitro laboratory tests for ataxia-telangiectasia.1 Rapidly dividing cells are the most vulnerable to anti-cancer treatments (this being, of course, the rationale for their use). This would suggest, in theory, a susceptibility for spermatogenesis in the postpubertal male (millions of cell divisions daily), along with a relative resistance in the prepubertal male child (male meiosis yet to commence) and in oögenesis from infancy through menopause in the female (cell division in suspension). In the male, the fact of a “blood-testis barrier” may offer a defense (Yauk et al. 2015); a selection against chromosomally imbalanced cells during gametogenesis may be another protective process. The direct assessment of gametic chromosomes offers insight. A number of studies have used FISH to detect structural and numerical chromosome abnormalities in the sperm of men during and after chemotherapy treatment. From these, we may draw some broad generalizations regarding time course and sperm safety. Tempest et al. (2008) studied men with testicular cancer treated with bleomycin-etoposide-cisplatin (BEP), and men with Hodgkin lymphoma treated with BEP: at six months after treatment, the frequencies of XY disomy and of nullisomy 13 and 21 were significantly increased. Aneuploidy frequencies declined at 18 months follow-up, though for some chromosomes remained elevated for up to 24 months. Rives et al. (2017) also studied sperm aneuploidy rate in a population of testicular cancer patients treated with either BEP chemotherapy or radiotherapy, and found a significant increase in sperm aneuploidy that mostly returned to baseline by 12 months (Figure 26–1). Hodgkin lymphoma can be treated with radiotherapy and chemotherapy with Novantrone-Oncovin-Velban-Prednisone (NOVP). Frias et al. (2020) observed post-treatment increases in aneuploidies of chromosomes X, Y, 18, and 21, but which returned to baseline by six months. These authors extrapolated their findings genome-wide and estimated that the proportion of sperm with structural or numerical abnormalities would increase from a baseline of 1.6% to 16%, noting that most of these abnormalities would be lethal in early embryogenesis. They concluded there to be no excess risks for pregnancies conceived six months after the completion of therapy. 1 Radiation and bleomycin, both having potent DNA-breaking properties, cause lymphocyte chromosome rearrangements in normal cells, and considerably more so in ataxia-telangiectasia cells. Gonadal Cytogenetic Damage from Exposure to Extrinsic Agents  797 In practice, it is to the experience of the “therapeutic experiments” of oncological medicine that we mostly appeal: the in vivo observations of those who have survived their cancer, recovered from their treatment, and have gone on to have or to attempt to have children. Have the children shown any excess of cytogenetic abnormality? Outcomes from five large studies are summarized in Table 26–1. Collectively, these report on more than 50,000 children of cancer survivors and in fact, show no difference Figure 26–1.  Sperm Chromosomes Pre and Post Cancer Treatments. Notes: Longitudinal data on sperm aneuploidy rate in a population of testicular cancer patients treated with either bleomycin-etoposide-cisplatin chemotherapy or radiotherapy. Sperm aneuploidy was assessed using FISH for chromosomes X, Y and 18. Source: Drawn from data in N Rives et al., Sperm aneuploidy after testicular cancer treatment: data from a prospective multicenter study performed within the French Centre d’Étude et de Conservation des Oeufs et du Sperme network, Fertil Steril 107:580–588.e1, 2017. Table 26–1.  Outcomes from Six Large Studies of the Offspring of Childhood Cancer Survivors SOURCES COUNTRY NO. OF OFFSPRING OF CANCER SURVIVORS CHROMOSOME ABNORMALITIES CLASS OF CONTROLS CASES (%) CONTROLS (%) Byrne et al. (1998), Meistrich and Byrne (2002) United States 2,198 0.20 0.10 Siblings Ståhl et al. (2011)* Denmark, Sweden 8,670 0.08 0.18 Population Signorello et al. (2012) United States 4,699 0.15 None Seppänen et al. (2016) Finland 6,862 0.06 0.07 Siblings Nielsen et al. (2018) Denmark 14,611 0.33 0.37 Siblings Jin et al. (2024)** South Korea 15,221 0.15 0.16 Population *Included only offspring of male cancer survivors; ** included only offspring of female cancer survivors.

798  NOXIOUS AGENTS between the prevalence of chromosome abnormalities2 in the offspring of cancer survivors when compared to the offspring of their cancer-free siblings or population controls. The prevalence of chromosome abnormalities is consistently ~1 in 600 regardless of the type of cancer, the age of cancer onset, the type and dosage of chemotherapy administered, and the use of radiotherapy. Data about miscarriage rates are in agreement: Green et al. (2009) followed up more than 4,000 pregnancies in which either the mother or the father was a cancer survivor, and they found no increase in the frequency of miscarriage in either group. SPECIFIC THERAPIES Chemotherapy. Lambertini et al. (2020) review the effects of various chemotherapy regimes on fertility, categorizing according to whether the risk of infertility is high, intermediate, low, or “unknown,” the latter applying to most of the newer targeted and immunological therapies (Helgadottir et al. 2024). In both males and females, the risk of treatment-related infertility is highest for alkylating agents, radiotherapy that includes the gonads, and conditioning chemotherapy prior to bone marrow transplantation. Patients with an interest in future fertility should be referred for oncofertility assessment prior to commencing treatment. Radiotherapy. Fertility is reduced in females who have had radiation therapy to the abdomen, and the risk of obstetric complication is increased. But their children appear to have no increased incidence of birth defects (Signorello et al. 2012; Seppänen et al. 2016). In the male, testicular radiotherapy causes a modest increase in sperm aneuploidy rate that returns to normal after 12 months (Figure 26–1). Ståhl et al. (2011) studied 2,488 children born one or more years after the diagnosis of testicular cancer in their father, most of whom would have been treated with radiotherapy, and there was no increase in congenital abnormalities in their children Radioisotopes. Radioiodine is used in thyroid cancer and may cause transient oligospermia and amenorrhea in males and females, respectively, but its use appears to be of no risk for miscarriage or for congenital abnormality (Clement et al. 2015). Infertility Associated with Cancer Therapy, and Prior Gamete Banking Preservation of gametes prior to treatment for cancer is a logical management, and sperm banking as “fertility insurance” for boys and men with cancer is now considered routine (Menon et al. 2009). The American Society of Clinical Oncology recommends that the risk of infertility, and options for fertility preservation, be discussed with all patients of reproductive age (and with parents or guardians of children and adolescents) before starting treatment (Oktay et al. 2018). All patients who express an interest in fertility preservation should be referred to a reproductive specialist. For adult men, sperm cryopreservation is the only established fertility preservation method and should be done before starting chemotherapy. Cryopreservation of testicular tissue, which does not require sexual maturity, remains experimental. For adult women, both embryo and 2 It is notable that these studies also detected no increase in the prevalence of non-chromosomal birth defects in the offspring of cancer survivors. Gonadal Cytogenetic Damage from Exposure to Extrinsic Agents  799 oöcyte cryopreservation are established methods or fertility preservation, while ovarian tissue cryopreservation remains experimental. Diagnostic Radiology Ionizing radiation is a well-known cause of cancer, and in theory could also cause gene mutations in the germ cells of exposed parents and lead to genetically mediated adverse outcomes in children inheriting the mutations. In practice, transgenerational effects of radiation have never been shown in human populations (Nakamura et al. 2023), and in general there is no convincing evidence for an increased risk of chromosome abnormalities in the offspring of parents who have been exposed to diagnostic radiology. A small effect may possibly exist for Down syndrome (DS) with respect to previous X-rays to the abdomen and pelvic area—that is, for X-rays in which the gonads may have been within or near the field of the film. In a study of 156 mothers and 149 fathers in whose DS children the “nondisjunctional parent” could be identified (using Q-banding polymorphisms), a history of X-ray exposure was more often recorded in older fathers and in younger mothers (Strigini et al. 1990). The odds ratio for the whole group was 1.85, although the lower limit of the 95% confidence interval was 1.0. If such an effect truly exists in younger mothers (and the statistics were borderline), it would seem that this slight influence becomes diluted as they get older and the age effect comes to be predominant. Nonmedical Exposures Radioactivity. The human germline may be relatively resistant to the damaging effects of radiation, compared with some animal models (Neel et al. 1990; Adriaens et al. 2009).3 The atomic bomb blasts at Hiroshima and Nagasaki in 1945 were not followed by a statistically significant difference in the rate of chromosome abnormalities in children subsequently conceived, according to a study commenced in 1967 (Nakamura 2006). The study population comprised 8,322 individuals born 1946–1972, age range at the time of study 12–38 years, one or both of whose parents were within 2,000 meters of the hypocenter “ATB” (at the time of the bomb), alongside a contemporaneous local control group of 7,976 who were either more than 2,500 meters from the hypocenter or not present in the city. Sex chromosomal abnormalities were seen in 2.28 per 1,000 in the former group and in 3.01 per 1,000 of the latter. The only instance of autosomal trisomy was a 15-year-old with standard trisomy DS, whose father had been exposed at Hiroshima. Given the structure of this study, deceased younger children and infants with autosomal trisomy were not included, although it is also to be noted that in separate analyses in Neel and Schull (1991), no significant correlation existed between parental exposure ATB and the frequency of stillbirth or congenital malformations. More 3 Of historic interest, a very early example of ill health due to radiation exposure is that of Marie Curie, who was awarded the Nobel Prize twice. One daughter of hers was a scientist, and she also won a Nobel Prize, and the other was a skilled pianist and gifted writer. A series of n = 2 is very small, but rather evidently there must have been normal chromosomal segregation in the meioses leading to these two daughters.

800  NOXIOUS AGENTS children of exposed parents had a small supernumerary abnormal chromosome than in controls (5 cf. 2, a difference not specifically commented upon in Awa et al. 1987). Of the balanced structural rearrangements, only two were confirmed as having arisen de novo (one each in the exposed and control groups). An earlier study with specific reference to clinically diagnosed DS in 9-month-old infants, undertaken during 1948– 1954 before the chromosomal basis of DS was known, had shown no increase among offspring of 5,582 exposed cf. 9,452 unexposed mothers, and indeed the figures were in the other direction (0.54 cf. 1.27 per 1,000) and despite the exposed mothers being on average slightly older (Schull and Neel 1962). The Chernobyl nuclear plant explosion occurred in April 1986, and a cloud of radioactivity was dispersed over Europe. With respect to DS, no subsequent rise in incidence was identified in a number of European countries apart from small clusters in Berlin and Belarus, the latter of interest in that the peak was confined to January 1987, nine months after the explosion (Little 1993; Zatsepin et al. 2007). In contrast, Bound et al. (1995) suggest a possible link between events in 1957 (a fire in a nuclear reactor) and the early 1960s (increased levels of fallout from nuclear testing) and peaks of DS prevalence in 1958 and 1963–1964 in the Fylde district of Lancashire, England. But by no means is a firm case made: post hoc does not necessarily mean propter hoc,4 and some fluctuation is normal. The same 1957 nuclear reactor accident had been proposed as the possible reason for a cluster of six cases of DS among the children of women who had attended the same high school in Dundalk, Ireland, during 1956–1957 when they would have been aged from 12 to 19 years. Of the 387 births to the former pupils from this period, the expectation would have been 0.69 children with DS. However, a stringent review of the evidence, including molecular analyses that showed one case to have arisen post-fertilization, led to the conclusion that in fact, chance alone was the probable basis for the “cluster” (Dean et al. 2000). While the germline, at least from the evidence outlined earlier, is apparently resistant, the same cannot be said for the bone marrow. Numerous studies on radiation exposure have shown that stable chromosome rearrangements may be induced, as measured on peripheral blood samples. Indeed, it is proposed that these changes can be used as reliable biomarkers of exposure. Populations in whom this effect has been seen include Russian nuclear plant workers (comparing those exposed to plutonium and those to gamma rays) from 1949 to 1989; New Zealand navy personnel who had served on ships during nuclear bomb testing in the Pacific Ocean in the late 1950s; American radiation technologists who had begun practicing before 1950 (ages at the time of study 71– 90 years); populations living near the Three Mile Island nuclear facility at the time of the 1979 accident; and even astronauts, unprotected by Earth’s atmosphere from solar radiation (Durante et al. 2003; Hande et al. 2003; Sigurdson et al. 2008; Wahab et al. 2008; Little et al. 2021). We are unaware of any evidence that individuals exposed in these ways might have acquired any gonadal damage and that their children could have been at risk for a chromosomal disorder. 4 Post hoc, ergo propter hoc (Latin) = Something happened after the event, and therefore it must have been due to the event.

Gonadal Cytogenetic Damage from Exposure to Extrinsic Agents  801 Industrial Agents. The male gonad is protected by the blood-testis barrier (Li et al. 2016), but it is prudent to imagine that the protection may not necessarily be absolute. Paternal occupation provides a surrogate marker for a variety of potential industrial agents. Olshan et al. (1989) assessed the father’s occupation for 1,000 DS children born in British Columbia from 1952 through 1973. Seven employment categories out of 59 showed odds ratios in the range 1.4–3.3, the lower confidence limit being not less than 0.9, in which exposure to various industrial agents could plausibly have occurred (including mechanics, janitors, metal workers, sawmill workers). But the increases in risk were small, and with 59 items under study there was of course the possibility of chance fluctuation. One category that might have seemed risky, namely “other chemical workers,” in fact had the lowest odds ratio of all (0.2). Exposure to volatile oil (as studied following clean-up after an oil spill at sea) may damage the marrow, with chromosomal breakages observed in peripheral blood, but there is no indication of damage at the level of the gonad (Francés et al. 2016). Given that most DS arises from maternal nondisjunction, maternal occupation may be more relevant. There may be an association between maternal socioeconomic status and meiosis II nondisjunction, suggesting that maternal occupational exposures might be relevant (Hunter et al. 2013). One study compared maternal occupation for 714 DS cases and 973 controls, and out of 19 occupational categories only one, “Life, Physical and Social Sciences” reached statistical significance, with an odds ratio of 4.57 (Keen et al. 2020). No specific occupational exposure could be discerned within this heterogeneous group, and the result may have arisen by chance. Pesticides have biological activity, and it is reasonable to raise a case that distribution across the blood-testis barrier might follow inhalation, absorption, or ingestion, and that the local effect upon gonadal tissue might be toxic. Giulioni et al. (2022) reviewed 64 studies correlating pesticide-exposure male fertility and noted that many pesticides have been associated with altered sperm count, sperm motility, and sperm morphology. Regarding sperm chromosomal abnormality, data are more limited: some studies did and some did not show an increase in chromosomal defects. Of the numerous agents, the strongest case could be made for carbaryl and fenvalerate in particular as potentially causative of autosomal and gonosomal aneuploidy, with sex chromosome disomy the most frequent single abnormality. The air we breathe, it is suggested, might convey mutagens in the form of industrial pollutants, and these might reach gonadal tissue (Somers and Cooper 2009). Farhi et al. (2014) found a borderline increased risk (odds ratio 1.14) for chromosome abnormalities in Israeli women exposed to high levels of air pollution in the first trimester, but these results were not confirmed by studies in China (Li et al. 2022b) and the United States (Ren et al. 2018), in which no association was seen between chromosome abnormalities and air pollution during the pre-conception period. In the male, Jurewicz et al. (2018) reviewed the impact of air pollution on a range of sperm parameters and found inconsistent results, with some studies suggesting an increase in sperm aneuploidy in men exposed to high air pollution and others finding no such difference. Landfill sites contain toxic matter, which might in theory contaminate the air in nearby residential areas; but in an analysis based upon more than 6,000 such sites throughout the United Kingdom and comparing populations living within and beyond 2 kilometers of these sites, no differences in the prevalence of DS were observed (Jarup et al. 2007). However, older mothers living within one mile of industrial sites from which solvent and

802  NOXIOUS AGENTS heavy metal emissions are vented may have a slightly increased risk for aneuploidies in their offspring (Brender et al. 2008). Confirmatory studies are needed. Bisphenol A (BPA), an estrogenic chemical used widely in plastic manufacture, has been shown to disrupt several different stages of oöcyte development in mice (Susiarjo et al. 2007). The fetal ovaries observed after pregnant mice were treated with low, environmentally-relevant doses of BPA during mid-gestation showed synaptic defects and increased levels of recombination. The mature females who were exposed as fetuses went on to have oöcytes and embryos with aneuploidies. There may be further environmental influences on the effect of BPA, in that variations in diet influenced the observation of meiotic abnormalities in exposed mice (Muhlhauser et al. 2009), thus demonstrating the complexities in studying environmental exposures, since even in laboratory animals it is practically impossible to keep all other variables constant. In a small study in humans, Lathi et al. (2014) found an increased risk of both euploid and aneuploid miscarriage in women with higher serum levels of BPA. In males, there is evidence that BPA decreases sperm quality, although the impact on fertility remains unclear (Presunto et al. 2023). Radwan et al. (2018) found a positive association between urinary concentrations of BPA and sperm sex chromosome disomy as measured by sperm FISH, although the proportion of abnormal sperm remained less than 2%. Reservations are already held concerning its use in human activity for other health-related reasons, and these data might be seen as one further reason for caution. Agent Orange (a mixture of phenoxylic herbicides) was used in the Vietnam War as a defoliant spray, and those exposed may have absorbed the chemical via the oral route in particular. A study of New Zealand Vietnam veterans some three or four decades after the war showed an increase in sister chromatid exchanges on blood samples (Rowland et al. 2007). Whether gonadal genetic damage results is controversial (Ngo et al. 2006; Schecter and Constable 2006; Fraser 2009); specifically, we are unaware of any evidence for an increased risk of chromosomal abnormalities in offspring. Recreational Agents. Tobacco smoking in mothers has been studied extensively in relation to the prevalence of DS. In a meta-analysis of 17 published studies, the overall relative risk in smokers compared with nonsmokers was 0.95, which was not statistically significant (Rudnicka et al. 2002). Nevertheless, a tentative role has been proposed for one particular mechanism: trisomy 21 due to nondisjunction in maternal meiosis II (MMII). In a case-control study in Atlanta, Georgia, cigarette smoking around the time of conception gave an odds ratio of 7.6 in mothers of MMII trisomic offspring compared with controls, in the <35-year age group (Yang et al. 1999). Very speculatively, smoking might diminish blood flow in the microvasculature of the perifollicular bed, and the resultant hypoxia could compromise some aspect of the oöcyte’s functioning as the meiotic process is reactivated. Concerning spermatogenesis, Beal et al. (2017) reviewed data from sperm FISH studies concluding that smoking likely induced a twofold increase in aneuploidy for all 22 autosomes and the sex chromosomes. Attempting to estimate the global impact of smoking, the authors assumed background livebirth rates for aneuploidies of paternal origin of 0.01% for autosomes and 0.075% for sex chromosomes and made the startling calculation that each year, smoking could be responsible for 160,000 extra cases of autosomal aneuploidy globally, and 1.2 million extra cases of sex chromosome aneuploidy. Secondhand smoke is difficult to assess, outside of controlled animal studies (Hung et al. 2009). A study of four adult male rhesus macaques exposed to secondhand smoke

Gonadal Cytogenetic Damage from Exposure to Extrinsic Agents  803 for six months showed no change in the X:Y ratio in sperm, which may indicate no increase in aneuploidy. Secondhand smoke–exposed pregnancies did not show increased DNA damage in their offspring as compared to babies born to nonsmoking mothers (de Assis et al. 2009). Alcohol is widely consumed. Saxov et al. (2023) reviewed studies seeking a link between maternal alcohol intake pre- and post-conception, and miscarriage. A small increased risk was not statistically significant. These authors speculate that pre-conception alcohol intake could possibly influence chromosomal integrity at oögenesis. Kaufman (1997), extrapolating from mouse studies, suggests that alcohol could disrupt components of the spindle mechanism as meiotic cell division proceeds in the ovum, leading to aneuploidy and, in that case, the untoward effect would operate prior to fertilization (in contrast to Fetal Alcohol Syndrome). In males, the observation in one study of a negative association between sperm disomy frequencies and alcohol consumption5 (Härkönen et al. 1999) should not at all lead one to advise that couples drink more heavily prior to a planned impregnation. Assessing alcohol and coffee intake, Torfs and Christianson (2000) determined the odds ratios, to have a child with DS, for “high” alcohol consumption (four or more drinks per week) was 0.54: in other words, a prima facie protective effect. High coffee consumption (four or more cups per day) appeared similarly protective, with odds ratio of 0.63. If these figures really did reflect biological reality, a possible mechanism would be a selective reduction in viability of a trisomic 21 as compared to a normal conceptus. GENETIC COUNSELING As Wyrobek and Adler (1996) commented three decades ago, “It has been more than half a century [1927] since Muller demonstrated that X-rays can induce germinal mutations in Drosophila, yet questions as basic as the existence of even a single human germinal mutagen remain unresolved.” McFadden and Friedman, writing in 1997, noted that no environmental agent has been identified in which it could be stated beyond reasonable doubt that this agent would cause chromosome abnormalities in the offspring of exposed parents. While some studies, as above, have shown increased rates of aneuploidy in sperm, the practical fact remains that no excess in children born with chromosomal syndromes has been observed. Only in 2001 could Marchetti et al. claim, with respect to their work on etoposide exposure with a mouse model, that “we know of no other report of an agent for which paternal exposure leads to an increased incidence of aneuploidy in the offspring.” Encouragement can be drawn from this largely negative information, and the counselor will usually be justified in offering substantially reassuring advice from the particular focus of chromosome abnormality. Reference to the commentaries earlier may provide useful supporting information for the individual agent of specific interest. Prenatal diagnosis would be a discretionary option, as would be preimplantation genetic testing for those whose treatment-related infertility required in vitro fertilization. A contrary view is put forward by DeMarini (2012), who argues that the failure to identify any human germ cell mutagens merely reflects methodological challenges of 5 But another study showed a positive association with alcohol, as well as with caffeine (Robbins et al. 1997).