Several different environmental chemicals including plastics, pesticides and pharmaceuticals have been shown to mimic or block estrogens and other endocrine compounds. Several of these chemicals disrupt reproductive development and cause infertility / reproductive disease in laboratory animals and occupationally exposed humans. These chemicals are thus referred to as Endocrine Disrupting Chemicals or EDC. For example, women (and laboratory animals) exposed in utero (in the womb) to high levels of the synthetic estrogen, DES, show reduced fertility, as well as, high levels of reproductive tract malformations and cancers (McLachlan et al. 2001). Gestational exposure to DES has been absolutely devastating for many DES daughters, where as others have experienced minor effects, implying individual variation in sensitivity to this environmental estrogen.
Prostate hypertrophy (enlargement) and prostate cancer are major problems in aging men. Gestational or neonatal exposure to estrogenic agents is thought to predispose males to prostate hypertrophy and cancer later in life. However, strains of laboratory animals differ in the effects of neonatal estrogen exposures on prostate and reproductive organ development. Sprague-Dawley strain rats have been reported to be more resistant than F344 strain rats to disruption of reproductive organ development by neonatal estrogen (Putz et al. 2001a; Putz et al. 2001b), and to prostate hypertrophy in aging males (Naslund et al. 1988).
Human epidemiological evidence has associated exposure to low-doses of a common drinking water disinfection by-product, bromodichloromethane (BDCM), with increased pregnancy loss (miscarriages) in humans (Waller et al. 1998). Exposure to 75 mg/kg BDCM during pregnancy resulted in full litter resorption in 62% of F344 rat litters, but 0% of SD rat litters (Bielmeier et al. 2001). SD rats were also insensitive to the induction of full litter resorption by an even higher dose of BDCM. SD and LE rats were more resistant than F344 rats to atrazine induced full-litter resorptions (Narotsky et al. 2001). Another strudy showed that SD and LE rats were not significantly affected, i.e., were insensitive, while F344 and Holtzman rats were sensitive to atrazine-induced embryo loss (Cummings et al. 2000). These observations raise concerns that Sprague-Dawley Rats may underestimate the effects of chemicals that cause miscarriage / pregnancy loss in unselected populations (US EPA White Paper on Strain/Stock/Species In Endocrine Disruptor Assays, 2003). Since many humans also show a high incidence of embryonic and pregnancy loss, the estimation of risk of pregnancy disruption in humans by environmental agents may be better modeled by strains that have not been selected for large litter size, and high, robust embryo survival like the SD derived CD rat.
The US Environmental Protection Agency (EPA) is preparing to conduct an Endocrine Disruptor Screening Program (EDSP) to screen chemicals for their ability to disrupt reproductive development and function. Of special concern is that the US EPA plans to use the Sprague-Dawley derived CD rat as THE predominant mammalian animal model for screening the effects of chemicals on reproductive development. A critical question is: will testing the safety of chemicals on a strain of rats that is more resistant to several estrogenic agents than certain other strains, and to the development of prostate hypertrophy, underestimate effects of such EDC on sensitive humans?
While data on human fertility is far from complete, recent estimates show considerable increases in impaired fecundity (commonly referred to as infertility) of reproductive age couples over the last several decades. A recent survey shows that 12% of the reproductive age population of the U.S. reported difficulty conceiving and/or carrying a pregnancy to term. That equates to about 7.3 million couples in the US with impaired fecundity. Advanced Assisted Reproductive Technologies (ART) are now used to induce about 100,000 ovulatory cycles and conceive 46,000, or one in 100, babies in the US. The economic costs of impaired fertility are staggering with a spending of about 2.9 billion dollars on infertility treatments in the US in 2002. Yet, even with such expensive ART, many couples are not conceiving and carrying a pregnancy to term, resulting in additional emotional costs. (As discussed on the Genetic Sensitivity to Fertility Drugs Web page) other women are hyperstimulated by fertility drug treatment and risk multiple pregnancy, premature delivery, and/or ovarian hyperstimulation syndrome.) Europe is showing an even higher incidence of impaired fecundity with more than 6% of all Danish children now being born following ART (Andersen and Erb, 2006). One of the reasons that many couples have to resort to ART is likely to involve exposure to a multitude of endocrine disrupting chemicals in the environment. Analysis of human tissue samples shows that humans have elevated body burdens of a large number of synthetic chemicals, many of which did not even exist before WWII. Unfortunately the effects of many of these chemicals and especially combinations of chemicals on reproductive development, function and health are poorly understood. So, inaddition to clear evidence that environmental exposures are disrupting reproductive development and function in wildlife species, there is increasing evidence for similar detrimental effects in humans.
Declining Sperm Counts: Concern has been raised that exposure to EDC maybe causing a decline in human sperm counts, semen quality and human fertility (Skakkebaek 2006) (Get PDF). Several epidemiological studies have shown a decline in human sperm counts in the US and especially in Europe over the last 50 years (Auger et al. 1995; Swan and Elkin 1999; Swan et al. 2000; Jorgensen et al. 2001). Others dispute whether sperm counts have actually declined over time, and suggest that apparent declines may be due to changes in sampling methodologies, etc. (Handelsman 2001; Safe 2004). Nevertheless, it is clear that testicular dysgenesis, testicular cancer and perhaps hypospadias and undescended testis are increasing in industrialized countries (Boisen et al. 2001; Skakkebaek et al. 2001; Safe 2004). These conditions as well as male infertility have been suggested to be part of a Testicular Dysgenesis syndrome (TDS) (Skakkebaek 2006). According to WHO standards, about 30% of young Danish men have semen quality in the sub-fertile range and over 10% in the infertile range Skakkebaek et al 2006. Furthermore, exposure to several environmental estrogens, including pesticides, herbicides, polychlorinated biphenyls and phthalate esters, and have been associated with decreased sperm counts and semen quality in humans (Rozati et al. 2002; Swan et al. 2003). Such pesticide and herbicide exposures have been hypothesized as explanations for the lower sperm counts and semen quality of men from mid-western farming regions than from urban regions of the US (Swan et al. 2003; Swan et al. 2003).
In October 2005, 40 experts in the toxicology and human fertility fields met and signed the Vallombrosa Consensus Statement on Environmental Contaminants and Human Fertility Compromise. (Get PDF). This statement recognizes the evidence for increasingly compromised human fertility and the involvement of EDC exposure. This consensus statement identified core points of scientific agreement, critical certainties and uncertainties, and important research priorities with regard to environmental reproductive health/fertility. Among several priorities, the Vallombrosa Consensus Statement recognizes the critical importance of determining the magnitude of genetic variation in susceptibility to environmental toxicants, as well as, which DNA polymorphisms within human, wildlife, and laboratory animal populations result in increased susceptibility to specific environmental contaminants. These DNA polymorphisms are often referred to as genetic markers. (For more information see http://www.healthandenvironment.org).
Our laboratory has long recognized that characterizing the genes controlling susceptibility to EDC is critical for understanding and preventing reproductive disease and infertility. We have identified and characterized well over16-fold genetic differences between strains of mice in susceptibility to the disruption of spermatogenesis, testes weight, and reproductive development by estrogen. The data reveal highly significant genotype x environmental interactions, e.g., strain-dependent dose responses, that confound the detection and prevention of environmentally induced infertility. These and other studies in mice and rats have raised concern that environmental estrogens may be disrupting reproductive development and reducing the fertility of sensitive individuals.
Endocrine Disruptor Screening Program (EDSP)
The US EPA is preparing to test a multitude of chemicals for their ability to disrupt reproductive development and function in the EDSP. Concern has been raised that the US EPA plans to use a strain of rats that is resistant to several Endocrine Disrupting Chemicals (EDC) as its main mammalian animal model in the EDSP (Spearow 2005). I (Jimmy Spearow) served as the sole external reviewer for the EPA's White Paper on Species/Stock/Strain on Endocrine Disruptor Assays. The purpose of this paper was to review the literature on species and strain differences in susceptibility to endocrine disruption, and to advise the EPA on the selection of appropriate mammalian animal models for the Endocrine Disruptor Screening Program (EDSP). Unfortunately, none of the errors I identified in my final reviewer’s critique were addressed in the EPA White Paper (Spearow 2003). Since this EPA White Paper (Parker and Tyl, 2003) was supposed to be peer reviewed, I prepared a Reviewer's Appendix to this EPA White Paper (Spearow 2005), which was finally released in 2005 with Congressional assistance. The White Paper and the Reviewer's Appendix are available: http://epa.gov/endo/pubs/program/whitepaper.htm
Since the EDSP will be used to help determine permissible environmental levels of EDC, choosing the most appropriate strain(s) is very important. Choosing a strain that is resistant to a given EDC as the animal model in EDSP assays risks underestimating the effects such EDC on normal and sensitive individuals, which could result in reproductive disease, infertility and population declines. Choosing overly sensitive strains will restrict commerce.
A major concern is that the Sprague-Dawley-derived CD rat, has been previously selected for large litter size, vigorous growth, and high resistance to arsenic trioxide. Not only are Sprague-Dawley rats are more resistant than F344 strain rats to disruption of reproductive development by estrogens (Inano et al. 1996; Putz et al. 2001b), by Bisphenol A (Steinmetz et al. 1997; Steinmetz et al. 1998; Long et al. 2000), and by lead (Apostoli et al. 1998; Dearth et al. 2004), they are also more resistant to the disruption of pregnancy by atrazine (Cummings et al. 2000; Narotsky et al. 2001), and bromodichloromethane (Bielmeier et al. 2001). These and many other strain-dependent susceptibilities are discussed in the Reviewer's Appendix.
The point is that lab animal strains (and humans) differ markedly in susceptibility to several, but not all, EDC and the most appropriate strains need to be selected for use in EDC screening assays so that effects on sensitive genotypes are not underestimated. Practices such as testing the toxicity of arsenic (Holson et al. 2000a), in a rat strain that has a history of selection for high resistance to arsenic (Poiley 1953; Lindsey 1979), raise questions as to whether toxic metal and oxidative stress-sensitive individuals will be adequately protected from chemicals with similar mechanisms of toxicity. Also problematic is testing the toxicity of Bisphenol A in a strain like the Sprague-Dawley derived CD rat which: 1) has been selected for large litter size (which tends to make it more resistant to estrogenic componds), and 2) has been shown to be much more resistant to BisPhenol A than the F344 rat at several reproductive developmental endpoints (Steinmetz et al. 1997; Steinmetz et al. 1998; Long et al. 2000).
Screening chemicals for endocrine disruptor activity using a rodent strain that is relatively resistant to the chemicals being tested is likely to: 1) underestimate the effects of such EDC on unselected individuals and populations; and, 2) result in the approval of higher environmental releases of EDC. This is likely to increase the risk of reproductive disease, infertility and population declines in sensitive populations and species.
Nevertheless, the US EPA has chozen to use the Sprague-Dawley derived CD rat as the predominant mammalian model for testing the safety of a multitude of chemicals in the endocrine disruptor screening program (EDSP). Some of the issues associated with the EPA's chosing a rat strain animal model that has been selected directly or indirectly for resistance to toxic metals and estrogenic compounds has recently been discussed in articles in The Dallas Morning News (Goetinck-Ambrose, 2007) (see http://www.dallasnews.com/sharedcontent/dws/news/healthscience/stories/052707dnentendocrine.3a08215.html and in Discover Magazine (Snell, 2009) ( see http://discovermagazine.com/2009/feb/22-very-tough-rat-big-risk-human-health )
Genetic Variation in Sensitivity to the Disruption of Sperm maturation and Reproductive Development by Estrogens
As published in Science, we have also discovered over 16-fold strain differences in sensitivity to the inhibition of spermatogenesis and testicular development by pubertal exposure to E2 (Spearow et al. 1999). (Get Abstract) (Please contact jlspearow@sbcglobal.net for a copy of the full paper). The linked figure shows the percentage of seminiferous tubules that are maturing sperm in the testis of CD-1 and C57BL/6J (B6) strain mice following a 3 week exposure of pubertal males to estrogen. The large litter size-selected mouse strain, CD-1, was highly resistant to increasing doses of estrogen and did not show a significant reduction percent of tubules maturing sperm, i.e. elongated spermatids, even in response to 40 µg Estrogen implants (Spearow et al, 1999). In contrast, even the lowest dose of 2.5 µg E2 markedly inhibited spermatogenesis in the unselected B6 strain mice. Testes of B6 mice exposed to 10 µg E2 and greater contained no sperm. Another strain of mice, C17, that had been developed as a randomly selected control also showed a marked reduction in sperm maturation in response to increasing doses of E2 (not shown) (Spearow et al., 1999). This study showed that large litter size seleced CD-1 strain mice were greater than16-fold more resistant than unselected B6 strain mice to the reduction in sperm maturation and tesis weight by estrogen.
These findings raised questions as to whether the large litter size selected animal models used for testing the safety of environmental chemicals would underestimate the effects of estrogens and xenoestrogens on normal, unselected individuals and populations.
We have now demonstrated much greater strain differences in sensitivity to the disruption of spermatogenesis and testicular development by E2 (Spearow, in preparation). We have also found that strains of mice differ in sensitivity to the induction of uterine weight and disruption of gestation by estrogen, and to the induction of uterine weight by the common xenoestrogen BisPhenol A. We have also found major differences between strains of mice in the ability of estrogen sensitized females to clear vaginal yeast (Candidia albicans) infections (Clemons et al. 2004). (Get paper).
EDC can disrupt estrogen signaling through diverse biological pathways including disregulating receptor binding and signal transduction, altering estrogen metabolism, and inhibiting steroid production (McLachlan et al. 2001; Newbold 2001; Song 2001; Kester et al. 2002). The finding that CD-1 strain mice have a much higher testicular estrogen sulfotransferase activity which is more resistant to reduction by estradiol than that of E2 sensitive B6 strain mice shows estrogen sulfotransferase as a mammalian genetic marker for SEDE (Spearow et al. 2001a) (Spearow et al. 2004)(Get Abstract) http://abstracts.co.allenpress.com/pweb/ssr2004/document/?ID=38611
In summary, these and many other studies demonstrate a large amount of genetic variation in susceptibility to the disruption of reproductive development and function by environmentally relevent endocrine chemicals. Will testing the safety of chemicals on a strain of laboratory animals that is more resistant to several environmental agents than certain other strains, underestimate effects of such EDC on unselected human populations?
Jimmy L. Spearow, Ph.D.
Email: jlspearow@sbcglobal.net (Please include "reproductive genetics" in the subject line).
Phone (530) 902-2041
Studies on genetic differences in susceptibility to endocinre disruptors by my laboratory were funded by the National Science Foundation. Integrative Biology and Neuroscience: Ecological and Evolutionary Physiology. Grant 9986077 Genetic Differences in Susceptibility to Endocrine Disruption. P.I. J.L. Spearow. 4/15/2000 to 4/14/2003.
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
Note that the information and opinions stated on this web site do not represent the position of the California EPA or the Department of Toxic Substances Control.
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