DIOXIN AND HUMAN SEX RATIO: THE SEVESO CASE

Paolo Mocarelli
University of Milano Bicocca
Department of Laboratory Medicine Hospital of Desio, Milan, Italy
Tel. +39 0362 383296/255
Fax +39 0362 383464
e-mail mocarelli@uds.unimib.it

The Seveso Case

Seveso is a small town with about 17000 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 40000 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 0.5-1 mL saved from about 30000 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 1976-79 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

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 fathers serum (12). The TCDD lowering sex ratio effect starts at
levels less than 20 ng/Kgbw. The mothers 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. Fathers serum TCDD levels less than 80 ppt were
a significant predictor of the probability of a male birth. Mothers 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 fathers 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.

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.

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: 2687 - 2695, 1986.

2.

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: 1559-66, 1990.

3.

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.
95-110. Cold Spring Harbor Laboratory Press, 1991.

4.

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 16:119-125,
1989.

5.

Mocarelli P, Marocchi A, Brambilla P, et al. Human data derived from the Seveso accident
relevance for human risk assessment. Toxic Subst J 12: 151-173, 1992.

6.

Mastroiacovo P, Spagnolo A, Marni E, et al. Birth defects in the Seveso area after TCDD
contamination. JAMA 259 : 1688-1695, 1988.

7.

Bertazzi PA, Consonni D, Bachetti S, et al. Health effects of Dioxin Exposure: A 20

Year Mortality Study. Am J Epidemiol 153: 1031-1044, 2001.

8.

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 2,3,7,8tetrachlorodibenzo-p-dioxin. Anal Chem 59: 2000-2005, 1987.

9.

Mocarelli P, Needham LL, Marocchi A, et al. Serum concentration of 2,3,7,8tetrachlorodibenzo-p-dioxin and test results from selected residents of Seveso, Italy.
J Toxicol Environ Health 32: 357-366, 1991.

10. Needham LL, Gerthoux PM, Patterson DG, et al. Serum dioxin levels in Seveso, Italy
population in 1976. Teratog Carcinog & Mutagen 17: 225-240, 1997
11.

Mocarelli P, Brambilla P, Gerthoux PM, et al. Change in sex ratio with exposure to dioxin.
The Lancet 348 : 409, 1996.

12. Mocarelli P, Gerthoux P M, Ferrari E, et al. Paternal concentrations of dioxin and sex ratio
offspring. The Lancet 355: 1858-1863, 2000.
13. Roman E, Peterson RE. Developmental male reproductive toxicology of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and PCBs. In: Korach KS, ed Reproductive and
developmental toxicology. New York: Marcel Dekker, 1998: 593-624.

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: 196-207, 1998.
15. Ryan JJ and Amirova Z Gender of children of russian chemical producers exposed to
dioxins. Organohalogen Compounds 53: 37-40, 2001.
16. Del Rio Gomez I et al. Prolonged effect on offsprings sex ratio following parents
ingestion of PCBs/PCDFs contaminated oil. Organohalogen Compounds 54: 315-319,
2001.