Combined Actions and Interactions of Chemicals in Mixtures

Furthermore, the estrogenic effects of simple mixtures of benzylbutylphtalate,
dibutylphtalate, and 17β-estradiol were tested in the yeast estrogen screen and the
activities were found to be approximately additive (Harris et al. 1997).
The complexity in evaluating reports on combination toxicology is illustrated by a
recent report on the interacting effects of phytoestrogens on the response to 17βestradiol
(Wang and Kurzer, 1998). At low concentrations, genestein and
coumestrol significantly enhanced estradiol-induced DNA synthesis, whereas at
high concentrations, inhibition was observed. Furthermore, differing effects were
observed with the other phytoestrogens. Thus, the type of interaction depended on
the concentration of the chemicals. This study deviates from many of the abovementioned
studies in being performed very thoroughly including dose-response
curves of the single compounds. These data could therefore form the basis for a
more detailed analysis of interaction effects.
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 acted 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.
Estrogenic activity in 2-, 3- and 4-component mixtures of o,p’-DDT, genistein, 4nonylphenol,
and 4-n-octylphenol were thoroughly evaluated in a yeast estrogen
screen (Payne et al., 2000). The models of dose-addition and response addition
were applied for calculation of the mixture effects and the conclusion was the
responses did not deviate from expected additivity. Both models were considered
as useful tools for evaluating the effects.
Another study from the same group investigated the impact of bisphenol A and
o,p’-DDT on the estradiol-induced estrogenic response in yeast cells (Rajapakse et
al. 2001). It was concluded that the combined effects did not deviate from
additivity and that the assumption that weak xenoestrogens are generally unable to
create an impact upon the already strong effects of endogenous steroidal estrogens
was not supported.
112