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Syzygy's Enlightenments
Developmental and Reproductive Toxicology Center of Expertise, Pfizer Global R&D, Eastern Point Rd, Groton, CT 06340
1 To whom correspondence should be addressed. E-mail: robert.e.chapin{at}pfizer.com.
Received May 17, 2007; accepted May 17, 2007
An observer standing on the Earth and watching the moon pass directly in front of the sun experiences a syzygy (the perfect straight-line configuration of three heavenly bodies). Now imagine that observer standing at a telescope viewing two distant stars, one behind the other. How does she know the further star is there, particularly if it is smaller than the nearer? The only way is to change the point of observation (i.e., induce a little parallax). Only by changing one's perspective can one determine whether an observation is truly one point, or two different points overlaid atop each other.
One challenge for observational astronomers, or course, is that the dimensions of their systems (solar or galactic) often require changes that are larger than a laboratory or even a planetary orbit. While biologists and toxicologists like to bemoan the complexity of our challenges, at least ours are not of that scale. But they are significant to us, and thus it is with special delight that we can enjoy the success of some of our peers when they come up with a new approach to an old problem, or, more gleefully for me, when they show that a tacit assumption in the field is false. For as our favorite bad-hair-life scientist said: "To raise new questions, new possibilities, to regard old problems from a new angle, requires creative imagination and marks real advance in science." (Albert Einstein). And when one's fellows do this in a way which is not only creative but thorough and careful, well, one can only sit back and murmur "(expletive deleted), I wish I had thought of that!".
We are treated to such a moment of joy with a paper in the June issue of Tox Sci by Kevin Gaido, Kam Johnson, and their colleagues at the CIIT Centers for Health Research, working together with Kim Boekelheide and long-time collaborator Sue Hall at Brown University (Gaido et al., 2007
).
A true superabundance of words has been penned about endocrine disruptors, so we will spare the gentle reader the long gory background here. Suffice it to say that when one treats a pregnant rat dam with an endocrine-active phthalate like di-butyl phthalate or di(2-ethylhexyl) phthalate (DEHP), the male offspring of that mother experience significant reductions in testosterone, as well as long-term problems with spermatogenesis, changes in Leydig cell structure within the testis, and in the testosterone-dependent ontogenic processes which set up the reproductive system for normal function for the rest of that animal's life (testis descent, brain structures, the epididymis, the prostate, and that external manifestation of baby males: a longer ano-genital distance). The changes in spermatogenesis begin almost immediately: the gonocytes, which give rise to the cells of spermatogenesis postnatally, become multinucleate within the developing seminiferous cords, and those cords develop into abnormal tubules. Because the baby male rats of DBP- or DEHP-treated mothers experience both the reduced testosterone and the altered inception of spermatogenesis and permanently deranged reproductive system, and because we know that spermatogenesis in adults depends on normal (that is to say, staggeringly high) intratesticular levels of testosterone (T), many people had assumed that the reduced T was responsible for the alterations within the seminiferous tubules.
My old professor derived immense joy from reminding us to test our assumptions... (though "reminding" is by far too gentle a word, but we will leave that to your imagination). We will simply note that when Gaido et al. tested this assumption by administering several different androgen receptor antagonists, they found that these all produced generally different profiles of gene changes (Mu et al., 2006). This then suggested that perhaps the changes inside the nascent seminiferous cords were not as dependent on the reduced T as people had thought.
Enter the mouse, escorted by Gaido et al., who brings with her a different place to stand and observe the stars. The pregnant mouse dam, when treated with DEHP, produces baby males with altered seminiferous cords and multinucleated gonocytes, but with no changes in T.
"Well", harrumphs the skeptic, "that's because the mouse metabolizes the phthalate faster than the rat and never generates the same blood levels!" Gaido et al. demonstrate that the mice have similar blood levels of active metabolite as the rats, and the kinetics of the blood levels are similar.
"Well" our skeptic continues, "the dose was still insufficient!" Gaido et al. tested many doses, including some which tread a significant way up the curve of maternal toxicity, and still show no reduction of fetal T levels.
"Well, that might be, but it only happens in that one strain of mouse, and if they had used a more appropriate strain, they would have found reduced testosterone!" Gaido et al. looked at both the C57 and C3H strains, and found the same lack of reduced T.
What's more, Gaido et al. look comprehensively at the genes associated with T production, and show a completely different pattern of response in the mice as compared with rats. Most interestingly, the testosterone syzygy is not the only altered alignment that is revealed by this new perspective: light is shed on many other genes whose suggested function based on rat data has been revised by these new data from the mouse. Surely there will be more manuscripts from this group as more postdocs dig through the reams of genomics data and generate dozens of new research hypotheses.
In fact, this paper is a good example of how toxicology ought to be done because it presents a deepening understanding of the basic biology along with appropriate analytics to make sure the dose formulations were correct, and to determine internal doses (i.e., blood levels), and it reports not just a single experiment (as is so often the case these days) but a rolling series of experiments. Thus, the reader receives a body of information that aggregates into real understanding.
So now we have a question: which species better models the human: the rat or the mouse? The immediate early response is to think about in vitro systems, for if one could show that short-term cultured fetal Leydig cells or testis pieces could replicate the reduced T secretion seen in vivo, then the stage would be set for determining whether this also happens in human tissue. Sadly, a recent report from Hallmark et al. (2007) suggests that this may not be quite so easy. If no one can get cultured cells to replicate the T reductions, then either we are collectively much less informed about culture requirements than we thought, or (less likely) it suggests that the fetal Leydig cells are responding to an extra-testicular influence. This would mean that the proximal target for a phthalate's androgen-lowering activity in the fetus is not the testis, which has implications for both toxicology AND for the basic understanding of fetal physiology and the developmental biology of the reproductive system, which is rendered much more complicated than we have previously thought (but is this any real surprise?). This may be yet another example of the complexities of everyday biology trumping our mental reductionism.
The lessons in all this? (1) "Test your assumptions," which I know makes my old professor cackle and slap his celestial desk with glee. (2) Spend the time digging through all those genomics tables; they hold significant insights. And finally, (3) pause and savor the particular intellectual joy afforded by a colleagues' creative and thorough problem-solving. Only they can take credit for it, but it inspires us all to think orthogonally and develop our own new points of perspective from which to discover our own syzygies.
REFERENCES
Gaido KW, Henslet JB, Liu D, Wallace DG, Borghoff S, Johnson KJ, Hall SH, Boekelheide K. Fetal mouse phthalate exposure shows that gonocyte multinucleation is not associated with decreased testicular testosterone. Toxicol. Sci. (2007) 97(2):491–503.
Hallmark N, Walker M, McKinnell C, Mahood IK, Scott H, Bayne R, Coutts S, Anderson RA, Greig I, Morris K, et al. Effects of monobutyl and di(n-butyl) phthalate in vitro on steroidogenesis and Leydig cell aggregation in fetal testis explants from the rat: Comparison with the effects in vivo in the fetal rat and neonatal marmoset and in vitro in the human. Environ. Health Perspect. (2007) 115:390–396.[Web of Science][Medline]
Mu X, Liu K, Kleymenova E, Sar M, Young SS, Gaido KW. Gene expression profiling of androgen receptor antagonists in the rat fetal testis reveals few common gene targets. J. Biochem. Mol. Toxicol. (2006) 20(1):7–17.[CrossRef][Web of Science][Medline]
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