Reproductive Effects of Male Dioxin Exposure
The Use of Offspring Sex Ratios to Detect Reproductive Effects of Male Exposure to Dioxins
Toppari et al. [EHP 104(suppl 4): 741-803(1996)], writing of the possible effects of dioxin, remark that no reproductive disorders in adults have been described after the accident in 1976 in Seveso, Italy. Publications since the submission of this paper put this assertion into perspective.
There is good evidence that the sex ratio (proportion male at birth) of mammalian (including human) offspring is partially controlled by the hormone levels of both parents around the time of conception (1). Egeland et al. (2) reported that men exposed to dioxin had low testosterone and high gonadotropin levels. Noting that this hormone profile is associated with female offspring, I predicted that exposure to dioxin would be associated with the subsequent births of excess daughters (3). This has since been confirmed with respect to the accident at Seveso (4) and to the workers exposed to wood preservatives contaminated by dioxin (5). The former study reported that a few heavily exposed parents produced a substantial and highly significant excess of daughters. The latter paper reported a slight but significant excess of daughters born to a large sample of men of whom it may be presumed were not severely exposed.
There are a number of illnesses and occupations in which men have been reported to sire an excess of daughters and to display low testosterone and/or high gonadotropin levels. The illnesses include non-Hodgkin's lymphoma and multiple sclerosis. The occupational exposures include those men involved in driving, diving, and those exposed to the nematocide dibromochloropropane (DBCP). Table 1 provides the references that substantiate the excess of daughters and the above hormone profile. Other occupational exposures that are associated with low offspring sex ratios are related to high-voltage installations (6-8), sodium borates (9), and carbon setting (10). Such exposures may be suspected of disrupting men's hormone levels, but I know of no direct evidence to document this suspicion. Thus, low offspring sex ratio may be regarded as a useful noninvasive monitor of reproductive hazards to men. Little is known about the effects of such exposures on women.
William H. James
The Galton Laboratory
University College
London, England
References
1. James WH. Evidence that mammalian sex ratios at birth are partially controlled by parental hormone levels at the time of conception. J Theor Biol 180:271-286(1996).
2. Egeland GM, Sweeney MH, Fingerhut MA, Willie KK, Schnorr TM, Halperin WE. Total serum testosterone and gonadotropins in workers exposed to dioxins. Am J Epidemiol 139:272-281 (1994).
3. James WH. Re: total serum testosterone and gonadotropins in workers exposed to dioxin [letter]. Am J Epidemiol 141:476-477 (1995).
4. Mocarelli P, Brambilla P, Gerthoux PM, Patterson DG, Needham LL. Change in sex ratio with exposure to dioxin. Lancet 348:409 (1996).
5. Dimich-Ward H, Hertzman C, Teschke K, Hershler R, Marion SA, Ostry A, Kelly S. Reproductive effects of parental exposure to chlorophenate wood preservatives in the sawmill industry. Scand J Work Environ Health 22:267-273 (1996).
6. Knave B, Gamberale F, Bergstrom EE, Birke E, Iregren A, Kolmodin-Hedman B, Wennberg A. Long-term exposure to electric fields: a cross-sectional epidemiologic investigation of occupationally exposed workers in high-voltage substations. Scand J Work Environ Health 5:115-125 (1979).
7. Kolodynski AA, Kolodynska VV. Motor and psychological functions of school children living in the area of the Skrunda radiolocation station in Latvia. Sci Total Environ 180:87-93 (1996).
8. Mubarak AAS, Mubarak AAS. Does high voltage electricity have an effect on the sex distribution of offspring? Hum Reprod 11:230-231 (1996).
9. James WH. Sex ratio of offspring of men exposed to sodium borates. Occup Environ Med 52:284 (1995).
10. Milham S. Unusual sex ratio of births to carbon setter fathers. Am J Ind Med 23:829-831 (1993).
11. Olsson H, Brandt L. Sex ratio in offspring of patients with non-Hodgkin lymphoma. N Engl J Med 306:367 (1982).
12. Olsson H. Epidemiological studies in malignant lymphoma with special reference to occupational exposure to organic solvents and to reproductive factors. [Ph.D. dissertation] University of Lund (1984).
13. James WH. The sex ratios of offspring of parents with multiple sclerosis. Neuroepidemiology 13:216-219 (1994)
14. Dickenson H, Parker L. Do alcohol and lead change the sex ratio? J Theor Biol 169:313 (1994)
15. James HW. The hypothesized hormonal control of mammalian sex ratio at birth-a second update. J Theor Biol 155:121-128 (1992).
16. Lyster WR. Altered sex ration in children of divers [letter]. Lancet 2:152 (1982).
17. Röckert HOE. Changes in the vascular bed in testes of rats exposed to air at 6 atmospheres absolute pressure. Int Res Commun Syst J Med Sci 5:107 (1977).
18. Röckert HOE, Haglid K. Reversible changes in the rate of DNA synthesis in the testes of rats after daily exposure to a hyperbaric environment of air. Int Res Commun Syst J Med Sci 11:531 (1983).
19. Potashnik G, Yanai-Inbar I. Dibromochloro-propane (DBCP): an 8-year re-evaluation of testicular function and reproductive performance. Fertil Steril 47:317-323 (1987)
20. Whorton D, Milby TH, Krauss RM, Stubbs HA. Testicular function in DBCP exposed workers. J Occup Med 21:161-166 (1979).
Response
In his letter, William H. James makes a valuable comment on our recent review on xenoestrogens (1) concerning reproductive effects of male exposure to dioxins. Whereas adverse reproductive effects of dioxin in laboratory animals are well documented (2), data on exposed human populations are scarce. The novel studies on offspring sex ratios of dioxin-exposed people are therefore very intriguing. Exposure to dioxins is associated with low male-female offspring ratio (3,4). Although this type of skewed sex ratio has been hypothesized to reflect impaired fertility, the underlying mechanisms have remained elusive. James (5) has suggested that parental hormone levels at the time of conception partially control mammalian sex ratio at birth. However, there is not enough experimental evidence to prove the hypothesis, and again, we do not have a theoretical basis for understanding the determination of sex ratio. Thus, both reproductive effects of dioxins and determination of sex ratio are research areas that need more of our attention.
Jorma Toppari
University of Turku
Turku, Finland
Niels E. Skakkebaek
Department of Growth and Reproduction
National University Hospital
Copenhagen, Denmark
References
1. Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette LJ, Jégou B, Jensen T, Jouannet P, Kreiding N, et al. Male reproductive health and environmental xenoestrogens. Environ Health Perspect 104(Suppl 4):741-803 (1996)
2. Peterson RE, Theobald HM, Kimmel GL. Developmental and reproductive toxicity of dioxins and related compounds: cross-species comparisons. Crit Rev Toxicol 23:283-335 (1993)
3. Mocarelli P, Brambilla P, Gerthoux PM, Pattersson DG, Needham LL. Change in sex ratio with exposure to dioxin. Lancet 348:409 (1996)
4. Dimich-Ward H, Herzman C, Teschke K, Hershler R, Marion SA, Ostry A, Kelly S. Reproductive effects of paternal exposure to chlorophenate wood preservatives in the sawmill industry. Scand J Work Environ Health 22:267-273 (1996)
5. James WH. Evidence that mammalian sex ratios at birth are partially controlled by parental hormone levels at the time of conception. J Theor Biol 180:271-286 (1996)
Mutagenic Carcinogens and Noncarcinogens in Transgenic Mice
In their recent article, Cunningham et al. [EHP 104(suppl 3): 683-686 (1996)] showed that in male B6C3F1 lacI transgenic mice after 90 days of dietary administration, 2,4-DAT (2,4-diaminotoluene) led to a twofold increase in mutant frequency (Mf), whereas 2,6-DAT did not. Based on this, the authors believe that these data help to validate the transgenic mouse model as a potential indicator of carcinogenic response and suggest that the Big Blue assay is also useful for mechanistic studies of carcinogenicity. We have doubts whether this belief is really supported by the compiled information, as can be easily derived from Table 1.
In our opinion the association of effects allows the following conclusions:
1. Both compounds are mutagenic in the in vitro S. typhimurium assay. Thus, both compounds can be considered equal with regard to their possible tumorigenic potential.
2. In the Big Blue assay, presence (for 2,4-DAT) or absence (for 2,6-DAT) of the association of proliferation, tumorigenesis, increase of mutation frequency, and UDS in the same tissue (liver) is recognized. However, whether the increase of the mutation frequency is just a sequela of the proliferative effect (clonic expansion of mutated cells that are also present in the negative control group) or a 2,4-DAT-induced tumorigenic DNA alteration remains unclear. In order to clarify this question, the effect of a pure proliferative stimulus (e.g., partial hepatectomy) should have been investigated in an additional group. Hence, it follows that liver tumorigenicity of 2,4-DAT might be causally linked either to its proliferative or its mutagenic effect or to both. Additionally, it is still questionable whether the described effect reflects a genuine mutagenic response, since the mutation frequencies induced by 2,4-DAT are quite similar after 30-day (Mf:9,3 x 10-5) and 90-day treatment (12,1 x 10-5). The statistical significance reported is mainly based on the lower spontaneous rate observed in the control group after 90-day treatment (Mf:5,7 x 10-5) in comparison to 30-day treatment (12,1x10-5). Thus, in our opinion, the Big Blue assay does not in this particular case provide more insight into the mechanism of tumorigenesis and its relation to mutagenesis.
Dr. P. Günzel
Dr. R. Reimann
Schering EG Institute for Experimental Toxicology
Berlin, Germany
Response
In their letter and table, Drs. Günzel and Reimann make three points concerning our recent publication [EHP 104(suppl 3):683-686 (1996)]. First, conclusion 1 states that compounds that are mutagenic in S. typhimurium should be "considered equal with regard to their possible tumorigenic potential." We believe there are few in the field of mutation research that would agree with this statement. Indeed, mechanistic research from our laboratories and others has focused on the overwhelming lack of concordance between in vitro tests and in vivo bioassays. Moreover, we feel that Drs. Günzel and Reimann illustrate the pressing need for in vivo models of mutagenicity that incorporate factors of physiological relevance such as chemically-induced cell proliferation. The Big Blue assay is such a model. Unfortunately, Drs. Günzel and Reimann misinterpret the data shown in their table. 2,6-DAT is not an hepatocarcinogen in mice or rats at the same or higher doses than proved hepatocarcinogenic for 2,4-DAT. Here the S. typhimurium was not useful in predicting potential carinogenicity. One must conclude that compounds that are mutagenic in S. typhimurium cannot be necessarily considered equal with regard to their possible tumorigenic potential. This is the point of evaluating chemicals using an in vivo mutation assay.
Second, Drs. Günzel and Reimann argue in conclusion 2 that the increase in observed mutant frequency (Mf) could arise from either 2,4-DAT induced cellular proliferation or from 2,4-DAT induced DNA damage. We agree. Further studies are necessary to define the mechanism whereby 2,4-DAT is mutagenic in the Big Blue assay. The point stressed in our manuscript, and the clear conclusion from our study, is that the carcinogen 2,4-DAT induces mutations in the liver, whereas the noncarcinogen 2,6-DAT does not.
Finally, Drs. Günzel and Reimann question the comparison of induced Mf data with aged matched controls. We are not comfortable making any other comparison. Indeed, we feel that the comparisons made by Drs. Günzel and Reimann in conclusion 2 clearly underscore the importance of including age-matched controls in any in vivo mutagenesis studies.
Thus, we remain confident that our studies with 2,4-DAT and 2,6-DAT using the Big Blue assay provide important insights regarding the relationship between mutagenesis and carcinogenesis. In addition, we believe that the development of the Big Blue and other mammalian in vivo mutation assays provide a significant opportunity for mechanistic studies regarding the role of induced mutations in the process of carcinogenesis.
Michael L. Cunningham
Kenneth R. Tindall
National Institute of Environmental Health Sciences
Research Triangle Park, North Carolina
Last Update: March 7, 1997