Combined Actions and Interactions of Chemicals in Mixtures

chance or experimental error. One way of dealing with this problem is to calculate
confidence intervals for the iso-effective doses of the single compounds and to add
a confidence belt to the line of additivity (Kortenkamp and Altenburger, 1998).
Kortenkamp and Altenburger (1998) have evaluated a study by Gaido et al. (1997)
applying the isobole method. The study focussed on binary mixtures of the
hydroxylated polychlorinated biphenyls, 2,4,6-trichloro-4-biphenylol and
2,3,4,5-tetrachloro-4-biphenylol, which had been analysed by Arnold et al. (1996).
The transcriptional activation of human estrogen receptors in human hepatoma
cells (HepG2) was determined. The cells were transfected with a reporter plasmid
containing an estrogen-responsive promoter linked to the luciferase gene. The
estrogen receptor was expressed from a second plasmid, referred to as estrogen
receptor expression plasmid. In order to study the influence of receptor
concentration on response, the cells were transformed with varying amounts of this
expression receptor plasmid (270, 27, and 2.7 ng plasmid per well).
Considering the experiments in which 27 ng plasmid was used, the evaluation
resulted in a data point for the mixture of the two hydroxylated polychlorinated
biphenyls which lied close to the additivity line. Thus, the hydroxy-PCBs act
additively which support the conclusion made by the authors. In contrast, the
results of experiments in which 270 ng expression plasmid per well were used for
cell transformations indicate that the combination effect of the same binary mixture
is synergistic. With the amount of expression plasmid further lowered to only 2.7
ng/well a strong antagonism between the two agents becomes apparent.
The work by Gaido et al. is interesting in that it shows how the type of
combination effect can change as the concentration at the target site, the estrogen
receptor, is varied. It is too early to suggest any explanations for this phenomenon,
but it would be worthwhile to pursue studies of this kind by measuring the effects
of combinations of the two hydroxy-PCBs at different mixture ratios.
7.5.5 Conclusion
Most of the work made within this field so far is based on in vitro experiments and
only very few in vivo experiments on mixtures on EDCs have been performed so
far. Such experiments will be one of the future challenges within the field of
endocrine disruption.
The majority of the -especially older- studies were inconclusive. Most of them
conclude that there appears to be additive effects, i.e. no interaction between
compounds is found, although a detailed mechanistic analysis has not been applied
in most cases. However, some studies, which showed absence of synergism, had in
fact overlooked synergistic effects as determined by a detailed evaluation of the
data. At least two studies, which involve the effect of hydroxy-PCBs on sex
reversal in turtle eggs and estrogen receptor activation, respectively, have shown
that synergism takes place.
However, the recent well-designed studies performed by Kortenkamp and coworkers
clearly show that the effects of estrogenic compounds do not deviate from
the expected additivity. In addition, additive effects of two antiandrogenic
compounds given in vivo were found.
Overall, most studies conclude that compounds are acting additively, so at the
present time there is no evidence pointing to the necessity of incorporating
synergism in the hazard assessment of weakly estrogenic chemical mixtures.
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