Why Methylmercury Remains a Conundrum 50 Years after Minamata
Department of Environmental Medicine, Environmental Health Sciences Center, and Center for Reproductive Epidemiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
1 For correspondence via fax: (585) 256-2591. E-mail: bernard_weiss{at}urmc.rochester.edu.
Received March 6, 2007; accepted March 7, 2007
Not too long ago, a common vision of economic vitality painted a landscape of factory chimneys pumping smoke plumes into the sky. We are now painfully aware of how flawed was our vision and of how the price for that vitality would come due only later. It stalked us in the form of widespread environmental contamination, with consequences for public health and welfare whose scope we still have not fully grasped. The Japanese coastal city of Minamata became a tragic model of how heedless industrialization could taint the lives of unwitting victims. These victims, largely fishermen and their families, began to experience a mysterious neurological affliction in the 1950's that eluded any known etiology. It eventually was traced to the consumption of seafood contaminated by methylmercury; its source the effluent inadvertently discharged by an acetaldehyde-manufacturing plant that used mercury as a catalyst in its production. Methylmercury had been known as a potent neurotoxicant for many years and its major clinical signs described in detail by Hunter and Russell (1940)
. That paper, in fact, served as the basis for the suggestion by Dr Douglas McAlpine, during a visit to Minamata for his studies of cerebral palsy, that this mystery disease ("kibyo") closely resembled methylmercury poisoning (Kavanagh, 2000).
Minamata provided the first clues to the extraordinary sensitivity of the fetal brain to methylmercury, a property more fully documented by an episode of methylmercury poisoning in Iraq in the winter of 1971–1972. This later catastrophe arose from the contamination of grain treated with a methylmercury fungicide (Cox et al., 1989). Minamata also established methylmercury as a model compound for the discipline that became to known as behavioral teratology (Spyker, 1975). Now, five decades and several thousand scientific papers later, Minamata disease is still a mystery of sorts; not the deep mystery of 1956 but not a superficial one either. The questions over which we struggle are different. They stem from how we reconcile laboratory science, epidemiological evidence, and environmental standards. Gross neurological deficits, the Minamata disease syndrome, have been replaced as markers of toxicity by delayed neurobehavioral development, as in Cox et al. (1989), and currently by performance on neuropsychological tests. These latter measures served as the source of the exposure standards recommended by the National Research Council (NRC, 2000) and then adopted by the United States Environmental Protection Agency. But these standards remain contentious, a result of the complex interplay of methylmercury neurotoxicity with factors such as nutrition and the presence of other neurotoxicants. Fish, the primary source of methylmercury for most populations, also provides nutrients such as docosohexanoic acid, essential for brain development. Aquatic species may also be contaminated by a score of neurotoxicants such as polychlorinated biphenyls (PCBs) and polybrominated diethyl ethers (PBDEs).
Such interpretive complexities continue to support an essential role for laboratory studies in which dose and exposure timing can be manipulated and in which life cycle stage and the functional and morphological end points examined are not amenable to observations in humans. These considerations led Onischenko et al. (2007) to study several outcomes in mice of early developmental exposure to methylmercury. Like many other research initiatives focused on neurobehavioral measures, their interests lay in the emergence of adverse effects beyond the period of early development and in the breadth of functions influenced by exposure. They also tried to extend their results mechanistically by examining a potential role for oxidative stress but this attempt yielded little information.
Their findings are consistent with what is currently known of how developmental exposure to methylmercury in experimental animals modifies behavioral performance later in life; at elevated exposure levels, exposed offspring tend to display performance deficits or aberrations. Placed in the context of the extensive methylmercury literature, their report also underscores the difficulties and complexities faced by investigators.
One such difficulty is diet, a problem that investigators from diverse areas have been forced to confront. For example, Boettger-Tong et al. (1998) viewed the problem from the vantage point of endocrine disruption and the phytoestrogens in most laboratory feed. In our own laboratory, we encountered methylmercury contamination, almost certainly due to the fish meal constituent in our chow that may have included tuna scraps (Weiss et al., 2005). Onischenko et al. (2007), on the basis of their tissue levels, seem to have sidestepped this problem, but it remains a threat that researchers need to be alert to.
Another question that those of us conducting mercury research have to weigh is when to conduct our tissue assays, brain in particular. In our own case (Stern et al., 2001), we selected postnatal day (PND) 4 for our mice because of the nature of our experimental design and its logistical requirements. Newland and Reile (1999) selected PND 1 for their rats, the optimal time, because it provides the best estimate of in utero exposure levels. Both laboratories found a sharp decline by PND 21 even though they had continued to expose lactating females. Sakamoto et al. (2002a), in rats, analyzed offspring brains at parturition and at PND 10. Over the course of that single period, brain levels dropped to about 20% of the original value, a phenomenon these researchers have also seen in nursing humans (Sakamoto et al., 2000b). Onischenko et al. (2007) may have underestimated in utero levels by choosing PND 7; extrapolating from Sakamoto et al. (2002a), their fetal brain levels, achieved by their target dose of 500 µg/kg per day, were likely closer to 4.0 µg/g rather than 0.9 µg/g. In the Newland and Reile (1999) study, mean brain levels at parturition came to 0.49 µg/g for their exposure level of 0.5 ppm in drinking water, which yielded approximately 40 µg/kg per day. Our own PND 4 brain values (Stern et al., 2001) were 5.62 µg/g in our high-dose 3 ppm group (about 0.6 mg/kg per day), which compares closely with the dose used by Onischenko et al., 2007). Our low-dose group, 1 ppm, yielded brain levels of about 1.6 µg/g. The logistics of exposure and assay make life complicated for toxicologists.
The most challenging choices experimenters confront are in the realm of behavioral end points. The possibilities are endless. Onischenko et al. (2007) selected three simple screening measures: locomotor activity, the rotarod, and the forced-swim test and two more complex end points, the Morris water maze and a newer instrument, an automated complex environment. Although they provided few details about the activity and rotarod devices, which yielded no methylmercury effects, these tend to be somewhat standardized. Nor did any effects on learning and spatial navigation emerge from the Morris maze testing. They did, however, find effects on the forced swim test. Both procedures deserve discussion.
The forced swim, or Porsolt test, is a screening test. Mice are dropped into a water-filled cylinder. At first, they tend to struggle but then become immobile. Although antidepressant drugs generally reduce immobility, it is neither a test for depression nor can it be called a model for depression. Although it has proven of some use in screening for antidepressant drugs, it does not mimic their therapeutic effects (Petit-Demouliere et al., 2005). After the initial test, for example, mice do not display their original behavior; on succeeding tests, all mice, treated or not, tend to remain immobile, a response that, in analogous contexts, has been termed, "learned helplessness." Swimming, however, could serve as another kind of end point. Spyker (1975), in her pioneering studies, found that in utero methylmercury exposure produced a variety of abnormal swimming positions in mouse offspring. Given the motor abnormalities associated with adult as well as with developmental exposures, it may prove useful, for future ventures, to record the behavior and analyze it with the kinds of quantitative methods that current technology makes available.
The Morris maze, like the Porsolt test, evokes neuroendocrine stress responses (Engelmann et al., 2006). It activates the hypothalamic-pituitary-adrenal axis and introduces another set of confounding variables for neurotoxicologists to contend with (as if we did not have enough already). Unlike most basic neuroscientists, we deliberately introduce additional complications by inserting toxic chemicals into the equation. We should try to keep unnecessary confounders to a minimum.
One of the measures employed by Ovischenko et al. (2007) introduces a different kind of confounding, one that deserves further exploration by neurotoxicologists. The arrangement they used to monitor the behavior of mouse groups resembles those used by biopsychologists to study the effects of what is termed "environmental enrichment." It refers to a situation in which the subjects live in groups with access to objects such as blocks, ladders, and running wheels. It provides an environment more consistent with that prevailing under natural conditions than does the typical laboratory cage. Over 40 years of research demonstrates that animals living in such an environment, especially during early development, exhibit increased cortical thickness, increased sizes of neuronal cell bodies and synaptic contact areas, increased numbers and extent and branching of dendrites, and more synapses per neuron than those raised in the standard environment (Weiss and Bellinger, 2006). They also show superior performance on a variety of behavioral measures, and some recent data indicate that they are resistant to the developmental neurotoxicity of lead. Moreover, such effects are visible even in adults and have triggered an explosion of research on their implications for the aging brain (Weiss, 2007). Whether such effects appeared in the mice studied by Ovischenko et al. (2007) would be a question worth pursuing.
Finally, those of us whose interests lie in how early developmental exposures unfold later in life need to balance several interwoven needs: how we choose end points, how we choose exposure levels and durations, when we choose to conduct chemical assays, and at which points during a subject's lifetime we choose to study functional outcomes. A useful model for how to balance all these factors comes from the studies of Newland and his coworkers. Newland and Reile (1999), referred to earlier, established dose parameters to encompass a practical range of exposures and brain levels. Newland and Rasmussen (2000) then showed how schedule-controlled operant behavior could be used to trace the consequences of developmental exposure as their rats aged. Newland et al. (2004) followed up by expanding the range of operant performances, demonstrating latent effects that appeared after 2 years of age. Another aspect of aging, and another kind of measure, appeared in the nonhuman primate studies by Rice (1998), who found impaired auditory sensitivity at 18 years of age following exposure from gestation to 4 years of age.
Ovischenko et al. (2007) have added to our understanding of how broad a swath of function responds to methylmercury exposure. What continues to surprise us is how much more there is still to learn.
ACKNOWLEDGMENTS
Preparation supported in part by grant ES013247 from National Institute of Environmental Health Sciences (NIEHS) to Bernard Weiss and by NIEHS Center grant ES01247 to the University of Rochester.
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