posted 27 October 2006
Andrade, AJM, SW Grande, CE Talsness, K Grote and I Chahoud. 2006. A dose–response study following in utero and lactational exposure to di-(2-ethylhexyl)-phthalate (DEHP): Non-monotonic dose–response and low dose effects on rat brain aromatase activity. Toxicology 227: 185-192.
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Decades of testing to set health standards-- what exposures are low enough to cause acceptable risk-- have been based on the assumption that high dose testing can predict low dose effects: Once the dose is low enough not to cause an effect, it is assumed that there is no need to test a lower dose. The lowest level tested with no effect is then used as a basis to establish safe standards for exposure to people.
This fundamental assumption for traditional toxicology has been challenged by lab experiments with animals that document "nonmonotonic" dose-response curves (NMDRC). These counter-intuitive patterns have low doses causing larger effects than higher doses. The key point is that high doses can't predict what happens with low dose exposures. This means that safety standards based on high dose experiments are invalid.
In this paper, Andrade et al. present one of the first examples of a NMRDC with phthalates. They show that lower doses of the phthalate DEHP suppress the activity of a key enzyme for masculinization of the male brain of rats, aromatase, while higher doses increase the enzyme's activity.
Context. To fully understand the importance of this paper it is helpful to bear in mind these background issues:
- A wealth of experimental studies with animals have documented impacts of DEHP on testicular function and developmental processes dependent upon androgen. These studies have typically used doses well above the ranges normally encountered by people. When Swan et al. (2005) found an association in baby boys between the mothers' phthalate levels and altered genital development, the effect was qualitatively consistent with animal results but appeared to be occurring at much lower exposure levels.
- Some experiments with cells indicate that DEHP also interferes with the action of the aromatase P450 enzyme, CYP19, which is necessary for the final step in converting androgen to estrogen.
- As the central nervous system of rats is developing, aromatase converts androgen to estrogen in local areas of the brain. Estrogen is necessary for sexual differentiation of key neural structures in specific brain regions. Without estrogen, male brains do not become masculinized.
- The standard approach in toxicological testing to set health standards is to use a limited number, usually 3 to 5, of high doses. Those experiments identify a level at which no effect can be seen, the 'no observed adverse effect level,' or NOAEL. Once the NOAEL is identified, an acceptable level for human exposure is calculated by using a series of safety factors that are supposed to take into account issues like differences in sensitivity among species, between people, kids vs. adults, etc. This approach assumes that dose-response curves are monotonic. It does not allow for 'inverted-U' or 'J' shaped dose-response curves, even though these non-monotonic curves appear regularly in studies of hormones, and with increasing regularity in studies of hormonally-active contaminants.
What did they do? Andrade et al. tested the effect of DEHP at two widely different levels of exposure: 0.015 to 1.215 and 5 to 405 mg/kg/day. The lowest dose was selected to match the estimated median daily intake of the general German population. The highest dose was selected to match a dose known to cause adverse reproductive effects in male offspring without causing overt harm in the mother. Hence the lowest dose used was 27,000 times lower than the highest dose.
Exposures were delivered to the pregnant female by daily gavage from day 6 during pregnancy to day 21 of lactation.
On Day 1 after birth (PND 1) and on Day 22 (PND 22), one or two pups of each sex and from each litter were selected for analysis of brain aromatase activity. They focused on a part of the prain, the hypothalamic/preoptic area (HPOA) that is known to be sexually dimorphic.
What did they find? They first confirmed, as expected, several basic facts about aromatase activity in the rat pups' brains:
- Aromatase activity in HPOA is higher just after birth (PND 1) than later (PND 22) in both males and females
- Aromatase activity is higher in HPOA than in the brain as a whole.
- Male aromatase activity is higher than female aromatase activity in HPOA on PND 1, but there is no difference on PND 22.
They then examined the impact of DEHP at different doses, in males vs. females, and on PND 1 vs PND 22.
||The most important finding was that in males on PND 1, high doses of DEHP increased aromatase activity while low doses suppressed it. The lowest dose to cause a significant decrease in aromatase was 0.135 mg/kg/day. At two intermediate doses, 1.215 mg/kg/day and 5 mg/kg/day there was no difference observed between exposed and unexposed animals. This dose-response curve follows a non-monotonic pattern.
In females on PND 1, DEHP had no statistically significant impact.
On PND 22, aromatase activity in males was enhanced by exposure to 0.405 mg/kg/day, one of the higher doses in the low dose range. No other doses, neither low nor high, caused a significant effect.
In contrast, aromatase activity in females on PND 22 was enhanced at all levels tested, including the lowest (0.015 mg/kg/day).
What does it mean? Andrade et al. have shown that DEHP alters the activity of aromatase in young rats following perinatal exposure. This activity is crucial for masculinization of the brain.
Most crucially, they show impacts at environmentally-relevant levels, far beneath the levels that have been tested following standard toxicological procedures. The pattern revealed in males on PND 1 was strikingly non-monotonic. They highlight the fact that "this biphasic response would have been overlooked if we had tested only the high dose range."
This finding means that a core assumption used to design toxicological tests of hormonally-active compounds is false. High dose tests do not predict low dose effects. According to Andrade et al. "Qualitatively different effects between low and high dose exposures may occur for several reasons, including saturation of biotransformation pathways or protein binding sites, depletion of intracellular cofactors and differences in ligand affinity and in efficacy of signal transduction" (see Welshons et al. (2003) for one in-depth exploration of this).
With that core assumption invalidated, there exists a strong likelihood that existing health standards for endocrine-disrupting compounds like DEHP are too weak. They have been set using high dose tests without study of possible low dose impacts. This is already clearly the case for bisphenol A, a compound that research has shown follow non-monotonic dose response curves in multiple endpoints measured.