DIOXIN AND HUMAN SEX RATIO: THE SEVESO CASE

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1 DIOXIN AND HUMAN SEX RATIO: THE SEVESO CASE Paolo Mocarelli University of Milano Bicocca Department of Laboratory Medicine Hospital of Desio, Milan, Italy Tel /255 Fax The Seveso Case Seveso is a small town with about inhabitants, between Lake Como and Milan, Italy. On Saturday July 10th, at 12:37, a cloud came out of a chimney at the small ICMESA factory (with about 170 workers) owned by the Geneve-based company Givaudan located on the outer-north outskirts of Seveso. This company produced intermediate compounds for the cosmetic and the pharmaceutical industries, including benzyl chloride and cyanide phenolacetic acid and 2,4,5 trichlorophenol (TCP) a toxic inflammable compound used as a bactericide and herbicide. It has been calculated that more than 1500 Kg of mixture went into the atmosphere including some Kg of pure 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD or dioxin). TCDD measuring technology 26 years ago was not as sensitive and reliable as today. There was no method or technology to measure TCDD in blood in those days and none accepted to donate adipose tissue to measure it so as to determine contamination. Therefore, the only measurement available was TCDD in soil and in dead animals. At the beginning, tracking of areas with dead animals and children with burns was also conducted. Exposure of people could be investigated only indirectly. A, B, and R zones were defined (1, 2) according to their decreasing dioxin contents, including about people. Information about effect of dioxin exposure in humans in Seveso have been published relating to chloracne metabolisms and cancer (3-7). In 1987 far away from Seveso, some scientists at Centers for Disease Control and Prevention (CDC, Atlanta, USA) were able to succeed to measure TCDD in human serum (8). We had ml saved from about samples and started a study. So far, almost 2000 samples have been measured and we are in a position to report the dose effect for many end points. In the future, results could be useful for risk assessment using human data. Data showing high levels of TCDD human exposure in Seveso have been published and other studies are in progress. High levels of TCDD did not induce liver cytotoxicity (this confirms our indirect previous observation) even after 20 years, they increased slightly in the lymphocyte number and the reaction to stimulation with PKW. The half-life of TCDD is much shorter in children than in adults (9, 10). TCDD and human sex ratio In recent years experimental and wild life contamination studies have shown that fetal and reproductive tissues are a very sensitive target of TCDD. We therefore have started studies on 1

2 TCDD effects on human reproductive system both in people exposed when young and in adults. We have described (11) for the first time a strikingly lower sex ratio at birth in the offspring of people highly exposed to TCDD. Researchers, had recently reported a reduced proportion of male births in the general population in several industrial countries and in selected populations such as in sawmill industry workers who were exposed to trichlorophenate contaminated with various dioxin congeners and those exposed to air pollution from incinerators. The possible origin (paternal, maternal or both) of the slight excess of female births was not clear. In view of this lack of knowledge and of the concern derived from the wide distribution of PCBs and PCDDs in the environment we have attempted to determine whether the parents sex and/or age at exposure in 1976 in Seveso affected the sex ratio of their children. We have measured the serum TCDD levels in exposed parents (239 males and 296 females) using serum samples drawn in 1976 and 1977 and have investigated the sex of their offspring (346 females, 328 males born from 1977 to 1996). Results show that there is an increased probability of female births (lower sex ratio) with increasing TCDD levels in the father s serum (12). The TCDD lowering sex ratio effect starts at levels less than 20 ng/kgbw. The mother s serum TCDD levels and the age at conception of either the father or mother were not predictors of outcome. Exposure of males during their preand puberty years may be especially relevant as fathers exposed when less than 19 years of age sired significantly more girls than boys. We demonstrated that serum dioxin levels lower than those already shown (11) may have a similar effect, if the exposure is to the father. Father s serum TCDD levels less than 80 ppt were a significant predictor of the probability of a male birth. Mother s serum TCDD levels were not a significant predictor of the probability of a male birth. Furthermore, exposure to males during their pre-or puberty years is linked to this sex ratio effect, as demonstrated by their offspring being predominantly female even though their offspring were conceived several years later when the father s serum dioxin levels were much less. This indicates that the pre-and pubertal years may be a very sensitive period to dioxin action in human males. Our data support the hypothesis of a permanent effect from the moment of exposure in males, who were exposed during their pre pubertal or pubertal years. This is evident by the excess of female newborns sired by these men even though they had dioxin levels less than 30 ppt, and even less than 15 ppt, at the time of conception of their offspring. We are not implying that we have evidence that serum dioxin levels of around 15 ppt lead to a decreased sex ratio. However, the evidence of this effect starts at initial TCDD levels lower than 80 ppt (lower than 16 ng/kgbw). The relationship of the median 1976 body burden level of dioxin (about 20 ng/kgbw, range= 3.5-3,960 ng/kgbw based on our serum measurements) in pre- and pubertal ages in males and the postpubertal effect on the sex ratio is in agreement with recent experimental animal data, which show a permanently altered sperm transit time through the epididymus (13)in adult rats exposed in utero and/or at lactation to mothers, who were dosed with TCDD at 64 ng/kgbw, and even at 25 ng/kgbw. 2

3 Recently, Bonduelle et al (14) observed that the sex ratio of children born from pregnancies after intracytoplasmic sperm injection (ICSI) with testicular or epididymal spermatozoa was modified to 0.66 and 1.46, respectively. Our finding of an altered sex ratio in the Seveso population is in agreement with the excess of females sired by fathers exposed to chlorophenates, which are known to contain traces of various dioxin congeners. Very recently, an excess of females has been observed from workers exposed to dioxin compared to a normal sex ratio in the city of Ufa (Russia) (15) as well in the YouCheng cohort people exposed to PCBs and PCDFs (16). We are now studying possible TCDD effects on quality of sperm. 3

4 References 1. Mocarelli P, Marocchi A, Brambilla P, et al. Clinical Laboratory Manifestation of Exposure to Dioxin in Children: a Five Years study of the Effects of an Environmental Disaster in Seveso, Italy. JAMA 256: , Di Domenico A, Cerlesi S, Ratti S. A two-exponential model to describe the vanishing trend of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the soil at Seveso, Northern Italy. Chemosphere 20: , Mocarelli P, Marocchi A, Brambilla P, et al. Effects of dioxin exposure in humans at Seveso, Italy. In: Banbury report 35: Biological Basis for Risk Assessment of Dioxins and Related Compounds.(Eds. Gallo, M.A., Scheuplein, R.J.and Van Der Heijden K.A.), pag Cold Spring Harbor Laboratory Press, Assennato G, Cervino D, Emmett EA, Longo G Merlo F. Follow-up of subjects who developed chloracne following TCDD exposure at Seveso. Am J Ind Med 16: , Mocarelli P, Marocchi A, Brambilla P, et al. Human data derived from the Seveso accident relevance for human risk assessment. Toxic Subst J 12: , Mastroiacovo P, Spagnolo A, Marni E, et al. Birth defects in the Seveso area after TCDD contamination. JAMA 259 : , Bertazzi PA, Consonni D, Bachetti S, et al. Health effects of Dioxin Exposure: A 20 Year Mortality Study. Am J Epidemiol 153: , Patterson DG Jr, et al. High resolution gas-chromatography/high-resolution mass spectrometric analysis of human serum on a whole weight and lipid basis for 2,3,7,8- tetrachlorodibenzo-p-dioxin. Anal Chem 59: , Mocarelli P, Needham LL, Marocchi A, et al. Serum concentration of 2,3,7,8- tetrachlorodibenzo-p-dioxin and test results from selected residents of Seveso, Italy. J Toxicol Environ Health 32: , Needham LL, Gerthoux PM, Patterson DG, et al. Serum dioxin levels in Seveso, Italy population in Teratog Carcinog & Mutagen 17: , Mocarelli P, Brambilla P, Gerthoux PM, et al. Change in sex ratio with exposure to dioxin. The Lancet 348 : 409, Mocarelli P, Gerthoux P M, Ferrari E, et al. Paternal concentrations of dioxin and sex ratio offspring. The Lancet 355: , Roman E, Peterson RE. Developmental male reproductive toxicology of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and PCBs. In: Korach KS, ed Reproductive and developmental toxicology. New York: Marcel Dekker, 1998:

5 14. Bonduelle M, Wilikens A, et al. A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Repr 13: , Ryan JJ and Amirova Z Gender of children of russian chemical producers exposed to dioxins. Organohalogen Compounds 53: 37-40, Del Rio Gomez I et al. Prolonged effect on offspring s sex ratio following parent s ingestion of PCBs/PCDFs contaminated oil. Organohalogen Compounds 54: ,