Ralph, JL, M-C Orgebin-Crist, J-J Lareyre and CC Nelson. 2003. Disruption of androgen regulation in the prostate by the environmental contaminant hexachlorobenzene. Environmental Health Perspectives doi:10.1289/ehp.5919

Prostate cancer strikes more men in the United States than any other cancer and it has become far more common a disease over the past several decades. While treatment has advanced significantly, reducing mortality rates, progress to understand the causes of prostate cancer have moved forward slowly.

This new paper opens up an important new front in the search for causes, demonstrating that the ubiquitous organochlorine contaminant hexachlorobenzene (HCB) disrupts normal development of the male reproductive tract by interfering with androgen action. Many other contaminants share the same mechanisms of action of HCB and thus are also implicated by these results.

In this paper, Ralph et al. use cell experiments in vitro and also experiments with mice to show that low levels of HCB enhance the responsiveness of prostate cells to androgen but that high levels of HCB suppress it.

What did they do? Ralph et al. carried out two different sets of experiments,

• first, working with prostate cells in vitro they varied exposure levels to HCB. The cells had been modified using standard molecular methods to produce a readily-detectable enzyme when the cell's androgen system was activated.
• second, they exposed male mice to HCB throughout gestation and lactation, and studied the effects of the exposures on different tissues, including the prostate and the epididymides. The mice also had been altered genetically so that androgen activation would be readily apparent and easy to localize to specific tissues. Three HCB dosage levels were used: low (5 mg/kg/day), medium (10 mg/kg/day) and high (20 mg/kg/day). [These doses... 5 - 20 parts per million), are far above the part per billion range used in the in vitro work and significantly above exposures that most people would experience.]

What did they find? In their in vitro work, they first established the normal pattern of responsiveness of the cells to dihydrotestosterone, or DHT.

With higher levels of DHT, Ralph et al. observe an increase in the activity level of their androgen response assay. The assay was based upon an gene that, when activated by an androgen, produced a unique protein, luciferase, that could be detected with great sensitivity.

Ralph et al. used this first graph to determine the amount of DHT to include in subsequent experiments: a level of DHT capable of producing half of the maximal response, which in the figure above can be seen to be approximately 2.5 nM DHT (it's a logarithmic scale).

They then ran a series of experiments varying the amount of HCB with that background level of DHT present.

The red line is the level of response obtained by 2.5 nM DHT without any HCB present. At levels of HCB exposure around 1 nM (parts per billion), Ralph et al. saw up to a doubling of the androgenic response in the presence of DHT. But at very high levels, the androgenic response was repressed.

This is a striking example of a "non-monotonic dose-response curve," in which low levels can produce larger effects than high dose levels.

Interestingly, Ralph et al.'s experiments also revealed that while the androgen receptor had to be present for HCB's effect to be observed, HCB itself did not bind to the androgen receptor. Its effect was being mediated via some other biochemical pathway.

Their work with mice involved two different parallel experiments, both with mice that had been altered genetically so that androgen activity could be readily observed and quantified. The first of these used a strain of mice which a novel reporter gene responsive to androgen within the prostate. The second experiment used mice with a reporter gene in the epididymides.

In addition to measuring the response of these reporter genes to different levels of HCB, Ralph et al. also measured prostate, liver, testes epididymides and overall body weights as well as other morphological characteristics, examined several tissues histologically, and measured serum hormone levels.

As suggested by the in vitro experiments, the effects of HCB were significant for a number of these variables, and in several low dose effects were the opposite of high dose effects. Ralph et al. conclude that their data indicate "that HCB agonized androgen action at low doses but antagonized it at high concentrations:"

There were no overt signs of toxicity from the exposure levels used. One of the strongest signs of overt toxicity caused by many contaminants at relatively high levels is weight loss. In contrast, the low and mid-dose animals in these experiments gained weight. Indeed, the low dose animals gained roughly 15% weight compared to controls.

Among many effects reported:

• Animals with medium and high levels of HCB exposure showed reductions in reporter gene activity within the prostate and the epididymides.
• Low dose treatments resulted in an increase in prostate weight compared to controls, while high doses reduced prostate weight.
• Low dose treatments increased the frequency of one marker for early sexual maturity whereas no effect was seen at the high dose level.
• Low dose treatment increased epididymides weight; no effect was induced by high dose treatment.

What does it mean? Ralph et al. conclude that their data provide:

Their results provide no conclusive links to prostate cancer. Rather, they reinforce the plausibility of links between exposure to this organochlorine and androgen-related development errors in mammals, demonstrably in mice, by extension in humans.

As Ralph et al. observe, androgens, specifically DHT, are profoundly important to the proper development of the male reproductive tract, including the prostate.

A contaminant that profoundly inteferes with normal control of that development, as their results compelling demonstrate for HCB, must necessarily be considered a candidate for prostate dysfunction in humans, especially given the widespread exposure of people to HCB. The challenge is that the experiments to prove these effects in humans would be unethical, and epidemiological studies remain fraught with many biases that increase the likelihood of false negatives.

Of additional concern is the fact that the standard treatment for prostate cancer is based on medical interventions to suppress androgen responsiveness in prostate tumors. If HCB enhances prostate androgen responsiveness in humans as it does in vitro and in mice, it may work against prostate cancer treatment.

Two patterns in their work were particularly interesting:

• low doses regularly caused effects at odds with high dose impacts. These low-dose effects undermine current methods used to protect public health from contaminants.
• while HCB was altering responsiveness to the androgen DHT, HCB did not bind directly with the androgen receptor. Ralph et al. propose that the response is mediated by HCB's known ability to interact with the AhR receptor.