Combined Actions and Interactions of Chemicals in Mixtures

dependent; eggs incubated at 26°C or 31°C develops into males or females,
respectively. Male-determined turtle eggs can be sex-reversed by applying estradiol
or estrogenic hydroxylated polychlorinated biphenyls (PCBs) to the eggs. In fact,
two of the hydroxy-PCBs (2’,4’,6’-trichloro-4-biphenylol and 2’,3’, 4’,6’tetrachloro-4-biphenylol)
in combination were effective in sex reversal at
concentrations that had little or no activity individually. Kortenkamp and
Altenburger (1998) reevaluated this study in detail. They applied the isobole
method, and concluded that the authors had correctly concluded that the two
compounds acted synergistically in affecting sex determination in turtle eggs.
These reports were followed by a paper reporting a dramatic synergistic effect on
estrogen receptor activation with combinations of environmental chemicals
(Arnold et al.1996), a paper that caused a great scientific, regulatory and public
concern around the world. Combinations of two weak environmental estrogens,
such as dieldrin, endosulfan or toxaphene (tech.mixt.) were tested in a yeast
estrogen system (YES) containing the human estrogen receptor and were reported
to act synergistically. The combinations were around 150-1500 times as potent in
estrogen receptor-mediated transactivation than any of the chemicals alone.
In the time after, several laboratories tried to reproduce the results without success.
Most remarkably was a report from a group of ten well-estimated scientists, who
tested the potential synergistic activity of dieldrin and toxaphene in ten different in
vitro or in vivo estrogen receptor assays (Ramamoorthy et al. 1997). The combined
activities of the two compounds were found to be essentially additive. The
compounds alone (up to 60 µmol/kg) or in equimolar concentrations were
investigated in 21-day old B6C3F1 mouse uterus and none of the treatments either
alone or in combination affected the uterine wet weight, peroxidase activity, or
progesterone receptor binding. In addition, the interaction of the other mixtures of
pesticides reported by Arnold (1996) was reinvestigated in two different yeastbased
assays and the results could not be confirmed. The story ended by a
retraction of the original paper by Arnold (McLachlan, 1997).
However, in 1997 a few other reports on synergistically interacting EDCs
appeared. Different combinations of the weak estrogenic compounds
benzylbutylphtalate, bisphenol A, p,p-DDE and 2,2’,3,3’,6,6’-hexachlorobiphenyl
were investigated in the MCF7 cell proliferation assay (Soto et al. 1997). Five
different combinations of three or four of these compounds induced what the
authors called a cumulative proliferation response. In this case no dose-response
curves of the single compounds were made and the conclusions were based on
intuitive effect summation.
Later Arnold et al. (1997) reported again on the synergistic interaction of
toxaphene, dieldrin and chlordane, this time with competitive binding to the
alligator and human estrogen receptor as the endpoint. Neither 220 nM chlordane,
630 nM dieldrin nor 200 nM toxaphene gave any binding response by themselves,
but in combination a response was observed. It was suggested that the estrogen
receptor might contain more than one site for binding environmental chemicals.
In contrast to these papers reporting synergism, endosulfan and dieldrin showed no
synergism in different estrogen receptor assays (Wade et al., 1997). Interaction was
tested with the estrogen receptor in or extracted from mammalian cells and no
synergism was observed. Furthermore, the compounds did not stimulate any
uterotrophic activity in vivo nor had any effect on pituitary prolactin or other
endocrine-related endpoints in immature female rats.
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