Our Stolen Futurea book by Theo Colborn, Dianne Dumanoski, and John Peterson Myers
 
 

 

Lehmann, KP, S Phillips, M Sar, PMD Foster, and KW Gaido. 2004. Dose-dependent alterations in gene expression and testosterone synthesis in the fetal testes of male rats exposed to di (n-butyl) phthalate. Toxicological Sciences,in press doi:10.1093/toxsci/kfh169 (available at http://dx.doi.org/).


Background on phthalates

In experiments exposing male rats in the womb to di (n-butyl) phthalate (DBP), Lehmann et al. found that low concentrations of DBP decrease the expression of genes critical to testosterone synthesis, at levels comparable to the maximum range of exposures experienced by the general public in the US.

The lowest level shown sufficient to alter gene expression, however, did not produce a measurable change in testosterone synthesis. That lowest level is also beneath exposures known to cause observable damage to the reproductive tract. At higher doses, both these effects are seen.

Changes were seen as low as 0.1 mg/kg/day. This is EPA's current estimate for a daily dose that is safe (the 'reference dose'). It is 4 orders of magnitude beneath EPA's current estimate for "no observed adverse effect level" based on mortality and 2 orders of magnitude beneath a NOAEL based on a study of reproductive tract abnormalities.

 

Context: Cholesterol is converted to the hormone testosterone by several enzymes, each of which is produced when specific genes are activated (expressed).

Testosterone itself is involved in the control of genes that, when activated, produce proteins necessary for normal development of the reproductive tract.

Thus decreases in expression of the genes making enzymes for chloresterol conversion will lead to lower testosterone levels, and subsequently to changes in the developmental processes under the influence of testosterone.

The specific impact will depend upon a variety of factors, including which gene(s)'s expression is altered, when and by how much.

What did they do?

Lehmann et al. treated pregnant rats with DBP by gavage during days 12 through 19 of gestation. The rats received DBP at several different levels, ranging from low levels --within the upper range of experienced by the US public-- up to relatively high levels --known to cause structural abnormalities in the sex organs of the male offspring of dosed pregnant females. These high levels still were not high enough to cause obvious adverse effects in the pregnant females.

On day 19, Lehmann et al. euthanized the male fetuses and performed a series of tests to search for differences between treated and untreated animals in the concentrations of specific molecules that are normally produced when the genes of interest are expressed. They examined both the mRNA associated with specific genes as well as the enzymes synthesized following gene expression.

What did they find?

Lehmann et al. found that that expression of certain key genes involved in making testosterone in the body was significantly reduced by exposure to DBP, even at low levels.

  These five graphs show changes in gene expression relative to normal (100%), one gene in each graph, for doses of DBP ranging from 0.01 to 1000 mg/kg/day. The farther below the 100% normal line, the greater the decrease in gene activity. Points falling significantly beneath normal are noted with the red asterisk.

 

In the graph above, 3ß-HSD, is suppressed at all DBP levels except 10 mg/kg. This gene is involved in converting cholesterol to testosterone

The c-Kit gene gene (left graph) follows a pattern quite similar to 3ß-HSD.

Both these genes are essential for normal proliferation an survival of spermatocytes. Expression of both were reduced by exposure to 0.1 mg/kg/day, which is close to levels of exposure that Americans experience today.

 

In the graph to the right SR-B1 parallels responses by c-Kit and 3ß-HSD, although no effect was seen at the lowest level used. SR-B1 mediates uptake of cholesterol by cells. While SR-B1 mRNA was reduced significantly at 1 mg/kg/day, the 20% reduction of SR-B1 protein induced at that exposure level was not statistically significant.

Each of these genes also displayed a non-monotonic response to increasing dose: lower and higher levels changed expression, while none showed a change at the intermediate range of 10 mg/kg/day.

 

Several other genes were only affected at higher doses of DBP.

  PBR, a gene involved in transporting cholesterol, was somewhat surprising: DBP increased PBR gene expression (as indicated by mRNA measurements, but reduced PBR protein in testicular interstitial cells. Lehmann et al. are not certain why this occurs but hypothesize that it could be due to either something that affects the protein after it is transcribed from mRNA (possibly caused by DBP) or differences in PBR gene expression in different areas of the testes.

 

 

DBP also suppressed expression of the gene Insl3 at higher doses (500 mg/kg/day). Insl3 previously has been shown to be involved in directing testicular descent. Mice without the gene show bilateral cryptorchidism.

DBP is known to cause cryptorchidism with exposures of 250 and 500 mg/kg/day. These results suggest that DBP's ability to cause cryptorchidism is a result of its impact on Insl3 activity.

Although effects of DBP on gene expression were evident at 0.1 mg/kg/day for some genes, decreases in testosterone concentration were evident only at exposures of 50 mg/kg/day.

What does this mean?

This study demonstrates that the ubiquitous contaminant DBP suppresses genes important in the development of the male reproductive system in rats, at levels lower than previously thought—levels comparable to current human exposure levels. It also sheds new light on how DBP disrupts the male reproductive system.

Lehmann et al. show that DBP reduces the expression of genes involved in testosterone synthesis in fetal rats, at levels below those causing structural changes to the male reproductive system. At higher levels, DBP causes cryptorchidism, retained nipples, and other reproductive abnormalities.

The current estimate by the US for DBP's "no observed adverse effect level" --or NOAEL-- is 125 mg/kg/day. That level is then used in risk assessment calculations to estimate a safe level for exposure, the 'reference dose,' which for DBP is calculated to be 0.1 mg/kg/day. Surveys by the US CDC already demonstrate that exposures to average citizens in the US can reach 0.113 mg/kg/day, i.e., above the reference dose.

Lehmann et al's results may add to pressure to revisit these guidelines. "Our results establish 50 mg/kg/day as a lowest-observable effect level (LOEL) and 10 mg/kg/day as a no-observable-effect-level (NOEL). How this unfolds will depend upon better understanding of what it means for gene expression to change significantly, at levels for which the endpoints studied (testosterone synthesis in the fetal testosterone synthesis, reproductive tract abnormalities) showed no adverse response. It is possible that these changes truly cause no adverse effect. On the other hand, it is also possible that measuring other endpoints under the control of the same genes, in these tissues or in others, would have shown adverse effects.

Lehmann et al. selected 10 mg/kg/day as the NOEL because it did not cause a statistically significant reduction in testosterone production. However, even lower levels (down to 0.1 mg/kg/day) caused statistically significant reductions in the expression of genes involved in several steps required for testosterone synthesis. The authors observe that the relevance of these alterations in gene expression at levels approaching human exposure levels “remain to be determined” and consider that they “may be sensitive indicators of DBP exposure but not necessarily of adverse consequences to DBP.” The authors also note that more powerful studies with larger numbers of animals and doses may be necessary to better understand the relationship between low levels of DBP exposure and effects on the development of the male reproductive system.

 

 
   
   

 

 

 

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