Archive for March 2011
The Dangers Of Using Simplistic Rules To Understand Complicated Processes Or ‘The Poison Is In The Dose’ Versus Reality
Posted March 13, 2011on:
Hello friends –
There is a lot of over simplification in discussions about autism on the Internet, sometimes I don’t think the people that use them really understand that their points are founded on primitive facsimiles of reality, but other times, I’m pretty sure they do know. That second group are the ones that really leave me in a confused rage; smart enough to know better (or have had the difference explained to them previously), but continue to rely on utilization of grade school quality parameters to govern complicated and entangled systems. It seems I’m often wrong when I wonder about the reason people do things (doh!), but when someone otherwise sufficiently knowledgeable relies on the crutch of simplicity because they think it bolsters their argument, I do tend to trust their motives before I consider human fallibility. It reminds me a lot of politicians, especially Republicans. [sorry]
That being said, one of the big simplifications you used to see a lot during the thimerosal wars was this gem:
“The poison in the dose.”
I googled this a bit. This phrase is attributed to Paracelsus, who Wikipedia tells me is considered ‘the father of toxicology’. He apparently wrote this:
All things are poison, and nothing is without poison; only the dose permits something not to be poisonous.
Good stuff. By the way, Paracelsus, who no doubt was pretty smart in his day, was born over six hundred goddamn years ago and the primary observation metrics available to Paracelsus was whether or not something died or not. Sure, oxygen is deadly in sufficient concentrations, as is water, salt, and everything else, so if we want to have a discussion that allows only for endpoints of livingness or death, the parameters laid out by him are good boundaries. However, if we would like our conversations to allow for somewhat more subtle changes associated with environmental exposures, something a dispassionate evaluation of the data dictates, we may need to find ways to have conversations that allow for endpoints other than death, and we will need to acknowledge that we have lots of evidence to suggest that there are inputs other than dose that are occasionally meaningful, no matter how this might affect our ability to take comfort in one study or the other. Even worse, we have actual, real empirical data to suggest there are times when there is an inverse dose relationship.
One of my pubmed alerts somewhat tangential to autism sent me the abstract for Differential mRNA expression of neuroimmunemarkers in the hippocampus of infant mice following toluene exposure during brain developmental period. It’s a doozy:
Toluene, a volatile organic compound with a wide range of industrial applications, can exert neurotoxic and immunotoxic effects. However, the effects of toluene exposure on developmental immunotoxicity in the brain have not yet been characterized. To investigate the susceptible window to toluene exposure during development and the effects of fetal and neonatal toluene exposure on the neuroimmune markers, gestational day (GD) 14 pregnant mice, postnatal day (PND) 2 and PND 8 male offspring were exposed to filtered air (control; 0 ppm), or 5 or 50 ppm toluene for 6 h per day for five consecutive days. The neuroimmune markers in the hippocampus of PND 21 were examined using a real-time RT-PCR and immunohistochemical analysis. Mice exposed to 50 ppm toluene on PND 2–6 showed significantly increased levels of nerve growth factor (NGF) and tumor necrosis factor (TNF)- mRNAs. In contrast, NGF and brain-derived neurotrophic factor (BDNF) and proinflammatory cytokines TNF-, CCL3, NF-kB, toll-like receptor (TLR)-4, astrocyte marker glial fibrillary acidic protein (GFAP), and microglia marker ionized calcium binding adapter molecule (Iba)-1 mRNAs were increased significantly in mice exposed to 5 ppm toluene on PND 8–12. These results indicate that low-level toluene exposure during the late postnatal period (PND 8–12) might induce neuroinflammatory mediators via TLR4-dependent NF-?B pathway in the hippocampus of PND 21 male mice. Among the three developmental phases, PND 8–12 seems to be most sensitive to toluene exposure. This is the first study to show developmental phase- and dose-specific changes in neuroimmune markers in infant mice following toluene exposure.
Essentially the authors took a bunch of mice exposed them to different amounts of airborne toluene at different days before and after birth, then looked for a variety of changes in immune system markers and neurotrophic factors in the hippocampus. Toluene was certainly capable of tinkering around with lots of systems that we know are skewed in the autism population. Curiously, what they found was that there were time dependent changes that had just as much of an impact than dose of toluene; and in fact, much, much lower doses of toluene were capable of causing more robust changes if the exposure occurred during critical developmental windows.
The authors state that the timeframe of exposures in this study, postnatal days 2 -6 and postnatal days 8 – 12 roughly map to the early and late third trimester of human fetal development, respectively. I’ve seen similar equivalencies in other papers, some with earlier and later timeframes, but certainly these timeframes are generally within the range that other papers have used. Consistent with the theme of this post, I’d just say that rat to human is difficult, and rat to human specific brain area and developmental timeframe equivalency is even more difficult.
The authors speculate that the difference in effect may be related to what was happening, developmentally within the brain at the time of toluene exposure that made the impact.
During this period, hippocampus undergoes an increase in size and a change in excitatory neurotransmission to allow for adult-like synaptic plasticity by the end of the second postnatal week (Dumas, 2005). This transiently heightened level of brain plasticity is shaped byenvironmental factors which have profound effects on this brain growth spurt (Goodlett et al., 1989). Furthermore, during this period, the immune system undergoes maturation to immunocompetence (Dietert et al., 2000).
There are also some stuff about why the hippocampus is a particularly promising target for investigation into effects of toluene exposure.
Here are a couple of graphs of their findings:
Check that shit out! During some very specific developmental timeframes, a decreased exposure resulted in increased physiological effect, not only that, the more affected animals received ten times less agent. Less poison, more effect. The exact opposite of what Paracelsus predicts. [Sorry for the formatting/stupid wordpress!]
Saliently towards autism, these graphs just happen to show some measurements that have great functional overlap with findings from autism. These graphs are for CCL3, an immune bugler of sorts, a chemokine, an agent responsible for attracting components of the immune response, one numeral down for CCL2, aka MCP-1, which we’ve also seen increased in the in autism brains, iba1, a marker for microglial activation, NGF and BDNF, neurotrophic factors that have a variety of signaling and maintenance processes in the CNS, and we have much data implicating altered BDNF levels in autism.
Not only did the authors observe an inverted dose relationship, some of the measurements found that the time dependencies are also reversed from what you might expect in that later exposure was worse than earlier exposure. Environmental exposures do not necessarily follow the linear timelines you might expect.
The idea of an inverted, or skewed dose relationship has actually been explored for some time. For example, The frequency of U-shaped dose responses in the toxicological literature
Hormesis has been defined as a dose-response relationship in which there is a stimulatory response at low doses, but an inhibitory response at high doses, resulting in a U- or inverted U-shaped dose response. To assess the proportion of studies satisfying criteria for evidence of hormesis, a database was created from published toxicological literature using rigorous a priori entry and evaluative criteria. One percent (195 out of 20,285) of the published articles contained 668 dose-response relationships that met the entry criteria. Subsequent application of evaluative criteria revealed that 245 (37% of 668) dose-response relationships from 86 articles (0.4% of 20,285) satisfied requirements for evidence of hormesis. Quantitative evaluation of false-positive and false-negative responses indicated that the data were not very susceptible to such influences. A complementary analysis of all dose responses assessed by hypothesis testing or distributional analyses, where the units of comparison were treatment doses below the NOAEL, revealed that of 1089 doses below the NOAEL, 213 (19.5%) satisfied statistical significance or distributional data evaluative criteria for hormesis, 869 (80%) did not differ from the control, and 7 (0.6%) displayed evidence of false-positive values. The 32.5-fold (19.5% vs 0.6%) greater occurrence of hormetic responses than a response of similar magnitude in the opposite (negative) direction strongly supports the nonrandom nature of hormetic responses. This study, which provides the first documentation of a data-derived frequency of hormetic responses in the toxicologically oriented literature, indicates that when the study design satisfies a priori criteria (i.e., a well-defined NOAEL, > or = 2 doses below the NOAEL, and the end point measured has the capacity to display either stimulatory or inhibitory responses), hormesis is frequently encountered and is broadly represented according to agent, model, and end point. These findings have broad-based implications for study design, risk assessment methods, and the establishment of optimal drug doses and suggest important evolutionarily adaptive strategies for dose-response relationships.
We have other examples from the synthetic world that may be of interest to autism. For example, in Developmental Exposure to Polychlorinated Biphenyls Interferes with Experience-Dependent Dendritic Plasticity and Ryanodine Receptor Expression in Weanling Rats the authors report an inverted dose relationship regarding exposure to PCBs and dendrite growth.
Developmental A1254 exposure significantly enhanced dendritic growth in cerebellar Purkinje cells and neocortical pyramidal neurons among P31 rats not trained in the Morris water maze, which is consistent with our previous observations that similar exposures accelerated dendritic growth in Purkinje cells and hippocampal CA1 pyramidal neurons between P21 and P60 (Lein et al. 2007). In Purkinje cells, this effect was observed among animals in the 1 mg but not 6 mg/kg/day A1254 group, whereas in neocortical neurons, responses were comparable between A1254 groups. The reason for the different dose–response relationship in different brain regions is not known. Possibilities include regional differences in RyR regulation (Berridge 2006; De Crescenzo et al. 2006; Hertle and Yeckel 2007) or differential upregulation of cytochrome P450 enzymes by AhR ligands in the cerebellum versus neo-cortex (Iba et al. 2003), which could result in regional differences in PCB toxicodynamics and toxicokinetics, respectively.
What about situations where we have evidence for an environmental factors already associated with autism? Neuroinflammation and behavioral abnormalities after neonatal terbutaline treatment in rats: implications for autism found that terbutaline administration at postnatal day 2 -5 resulted in chronically activated microglia and behavioral abnormalities in rodents, but the same dose in days 11 – 14 resulted in no such effect. Same dose, different time, different outcome.
There is more, lots more, but how many times must a rule fail primitive logical tests before we acknowledge that it’s utility in complex discussions is extremely limited? This absolutely is not meant as an expose meant to reignite discussions about thimerosal, but rather, to illustrate the dangers of trying to understand complicated rules by leveraging simplistic heuristics. There’s a lot of that in the autism discussion landscape; it is a dangerous concoction of hubris and faith to think that we can have gain meaningful insight into our shared mystery by applying very simple rules.
I haven’t seen the ‘poison is in the dose’ canard used for a while now. Good riddance and long live models that are not exceedingly simple to invalidate.
Elegant Observations, Feedback Loops Within Entangled Systems, and Possible Clues On Why So Many People With Autism Are Male – “Sex Hormones in Autism: Androgens and Estrogens Differentially and Reciprocally Regulate RORA, a Novel Candidate Gene for Autism”
Posted March 8, 2011on:
Hello friends –
Whatever your take on the predominant cause of autism(s), your thoughts on the appropriateness, or inappropriateness of research allocations in the autism realm, one thing can’t be denied; collectively, a lot of researcher time and dollars have been poured into autism research. We’ve learned some important things, some cool things, some confusing things, some obscure things, and some useless things. But even with all of the resources we have applied towards understanding autism, one of the most curious unknowns is also one obvious to the most rudimentary observations, the persistently skewed male to female ratio, with a finding of three or four males to every female with autism. It is one of the most vexing questions, almost taunting us with seeming obviousness, but consistently elusive. It isn’t just autism, lots of other neurological conditions are similarly tilted, and in a bazillion animal models it seems an unfortunate fact that being born male simple predisposes you (or rat you) to a variety of things you’d rather not have.
The ideas I have seen floated most frequently to explain this observation involve the effect of prenatal testosterone and the associated ‘extreme male brain’ theory, a loss of genetic backups to compensate for mixups, and synergistic effects of testosterone on chemicals, notably, mercury; explanations which I generally like a little, a little less, and almost not at all, respectively. An idea I’ve floated a couple of times, but that seemed even less accepted (or, more likely, completely unnoticed), was that estrogen might be acting in a protective manner; as it is known to exhibit attenuate the effects of neuroinflammation and oxidative stress. Indeed, it is starting to look like estrogen receptors are expressed in a large variety of situations salient to CNS processes.
A few weeks ago, the same group that has published some immensely cool studies on epigenetics and brain proteins, genetic expression differentials within twin siblings with autism, and circadian rhythm alterations comes a paper which may give us insight into this question, so that is pretty exciting. Even cooler, it invokes a negative feedback loop in a complicated system and is built upon the foundation of several earlier studies on a protein implicated in lots of things we know are awry in autism. That study is Sex Hormones in Autism: Androgens and Estrogens Differentially and Reciprocally Regulate RORA, a Novel Candidate Gene for Autism (full paper). Here is the abstract:
Autism, a pervasive neurodevelopmental disorder manifested by deficits in social behavior and interpersonal communication, and by stereotyped, repetitive behaviors, is inexplicably biased towards males by a ratio of ~4:1, with no clear understanding of whether or how the sex hormones may play a role in autism susceptibility. Here, we show that male and female hormones differentially regulate the expression of a novel autism candidate gene, retinoic acid-related orphan receptor-alpha (RORA) in a neuronal cell line, SH-SY5Y. In addition, we demonstrate that RORA transcriptionally regulates aromatase, an enzyme that converts testosterone to estrogen. We further show that aromatase protein is significantly reduced in the frontal cortex of autistic subjects relative to sex- and age-matched controls, and is strongly correlated with RORA protein levels in the brain. These results indicate that RORA has the potential to be under both negative and positive feedback regulation by male and female hormones, respectively, through one of its transcriptional targets, aromatase, and further suggest a mechanism for introducing sex bias in autism.
The press release and google news cycle for this paper seemed to have been well ahead of the pubmed robot; Kev had a post on this study a few weeks before it hit pubmed with a postdate. I generally skip out on the interest story/vaccine fairytale story/vaccine nightmare story/lost child nightmare story that is the google news autism feed, but in this case, it harbored a story on a paper that I was actually interested in. In any situation, the paper landed in pubmed this morning, and is available in full via PLOS, so great stuff is available to us all.
The paper starts with some of the backstory, the ‘inexplicable’ male predominance in autism, some of the theories on why this might be the case, and most importantly, details on previous findings by this set of researchers on reduced levels of RORA in the CNS of people with autism, a protein with a great number of functions of interest to the autism community.
Together, these results link molecular changes in RORA in peripheral cells to molecular pathology in the brain of autistic individuals. These findings are particularly relevant to ASD as RORA is involved in several key processes negatively impacted in autism, including Purkinje cell differentiation, cerebellar development, protection of neurons against oxidative stress, suppression of inflammation, and regulation of circadian rhythm. Behavioral studies on the RORA-deficient staggerer (RORA+/sg) mouse, primarily used as a model to study ataxia and dystonia, further show that RORA is also associated with restricted behaviors reminiscent of ASD, such as perseverative tendencies, limited maze patrolling, anomalous object exploration as well as deficits in spatial learning.
It’s tough to find a protein with a greater key word hitlist for our population of interest than Purkinje cell differentiation, cerebellar development, protection from oxidative stress, suppression of inflammation, and regulation of the circadian rhythm. In fact, I’d be shocked to find a protein touching so many fracture points that wasn’t found altered in the autism population; it makes too much sense within the framework of an entangled system and what we already know about the physiology of autism. Remember that the previous paper found decreased RORA in the brain of people with autism; i.e., less of a protein that protects from oxidative stress, supports Purkinje cell development, and suppresses the inflammatory response. A relative lack of RORA makes a depressingly good amount of sense.
That being said, what makes the current paper so interesting is that they found the RORA is differentially, and inversely modulated by female and male hormones (i.e., testosterone and estrogen). But even more insidiously, one of the downstream products regulated by RORA, aromatase, participates in the cleavage of testosterone to estrogen; the authors essentially describe a negative feedback loop. It turns out, not only is RORA decreased in the CNS of autism, but so too is aromatase.
We also show that one of the transcriptional targets of RORA is aromatase, which is a crucial enzyme in the biosynthesis of estrogen from testosterone. It is noteworthy that both RORA and aromatase proteins are decreased in the frontal cortex of autistic subjects, and that the level of aromatase protein is strongly correlated with the level of RORA protein in the brain tissues. We therefore propose that the reduction of RORA observed in autism is exacerbated by a negative feedback mechanism involving decreased aromatase level, which further causes accumulation of its substrate, testosterone, and reduction of its product, estradiol. Testosterone and estradiol respectively exhibit negative and positive feedback regulation of RORA expression as illustrated in Fig. 5, which summarizes the principal findings of this study. Thus, a deficiency in RORA in autistic brain is expected to be further aggravated by increased levels of testosterone due to suppression of aromatase, a transcriptional target of RORA.
This is pretty neat; it shows how simply being male can lead to the downregulation of a system with tendrils attached to a great number downstream processes we know to be disturbed in autism.
I particularly liked that this paper established a chain of learning more, something I think we can all agree is a great idea. Some of the people on this study have been plugging away with some interesting ideas for a while, all of which, I believe, are ancestors of these findings. They had two really neat papers on genetic expression in autism twins with differential degrees of autism severity, both of which used genomic bioinformatic tools to understand which the genetic pathways were affected. This is actually rather brilliant; they essentially leveraged the genetic uniqueness of the twins to gain more insight into which processes were being affected in autism by seeing which genes were differentially expressed in identical twins that manifested differently, using genetics to learn about what is happening a layer above the genome. Next, the original RORA paper began to probe the mechanism by which the previously observed expression was achieved, they found that a particular protein, RORA, was overmethylated and consequently at depressed levels. Another bioinformatic approach told them that RORA was a particularly attractive candidate for further evaluation based on its descendant interactions, and the association between RORA, aromatase, and sexual hormones appeared. Beautiful.
All that coolness not withstanding, some of the articles I saw on this lacked the caution and nuance we ought to see with these kinds of findings; the paper was pretty clear that previous CNS studies hadn’t shown decreased RORA in all of their samples, just most of them. This doesn’t answer all of our questions about the male dominance of autism, but we do know more than when this study was published, and that is pretty cool. Hooray for knowledge.