The honor of being the first recipient of our 'worst of scienceblogs.com' award goes to revere, the collective sobriquet of some self-described, anonymous 'senior public health scientists and practitioners'. Their winning post Bisphenol A. What's all the noise about? wins not only for its content, but for the near rabid way the blogger(s), revere, responds to scientific challenge.
First, a little background. Some time ago, the hypothesis was advanced that big, hydrophobic (i.e. greasy) molecules, of about the right shape, and especially if they contained polarizable atoms such as chlorine, might bind to steroid receptors. Steroids, like the sex hormones estrogen and testosterone, are themselves hydrophobic, so the cell receptors that detect these hormones are hydrophobic. 'Xenestrogens' -- molecules like bisphenol A and dieldrin, a pesticide -- don't fit the shape of the steroid binding site, but they do have the right electrostatic properties. So, it was hypothesized, those receptors might either be turned on by the xenestrogens (agonism), or the xenestrogens might block the real hormones from acting on the receptors without turning the receptors on themselves (antagonism). And alterations in sex hormone response are obviously bad for adults, but they're even worse for fetuses and infants, causing birth defects and developmental disorders. I'll post elsewhere why Greenpeace and their environmentalist fringe cohorts embraced the xenestrogen theory like a harpooned, dying whale; here I'll just point out that it's on its face a plausible, testable, and perfectly scientific hypothesis.
Problem is in the numbers. When people measured the binding constants of bisphenol A (BPA) to the nuclear estrogen alpha and beta receptors, they found it binds about 100 times less well than estrogen, its real target. More seriously, the binding constants for BPA are around 1 micromolar (1 uM). That means, if you expose the receptor to a 1 uM solution of BPA, half the sites will be occupied -- either turned on weakly, or blocked. But the highest measured levels of BPA in human plasma are more like 0.01 uM, or about 100 times lower than the binding constant. At that level, less than 1% of the sites are occupied, and the chances of a physiologically relevant effect are zero.
OK, plausible hypothesis refuted, right? Well, not if your scientific career depends on the hypothesis. Within the last few years, it has been proposed that estrogen receptors on the cell membrane respond to xenestrogens at much, much lower levels. If that were so, it would rescue the entire xenestrogen hypothesis, and raise significant concerns about the levels of BPA in the environment. BPA, by the way, is a building block of the polymer polycarbonate, an incredibly useful plastic because of its clarity and toughness, used in everything from the lenses of my eyeglasses, to baby bottles. Polycarbonate is a polymeric carbonic ester of BPA; in its manufacture, a little BPA remains in the polymer, but also the links between BPA and the carbonate groups can slowly be degraded by exposure to water, leading to release of BPA.
Revere's post ont he subject is really vague on specifics; so I asked him via a comment, how he reconciled the binding constant data with the reported biological effects. I was surprised, to say the least, by the response. Instead of answering, he answered with ridicule:
Mr. Harbison: You're right. Nothing designed like a bumblee could possibly fly. But it does. The literature showing effects at environmental levels is getting quite large. But you're safe. It's impossible! LOL.
I responded
The epidemiological literature showing effects of EM fields on health is also quite large. Nonetheless, physicists discount them, because of lack of a feasible mechanism. You, on the other hand, have postulated a mechanism, and that mechanism is disruption of the endocrine system. In fact, the people who are pushing bisphenol A as an ED (endocrine disruptor: RWP) have volunteered the estrogen receptor for the job. The biochemistry of binding of small molecules to hormone receptors is well known. And the numbers don't add up. Unless you can provide me with a receptor with a dissociation constant for bisphenol-A in the neighborhood of 10 nM, you're in the same category as the EMF crowd. The antagonism of estrogens will be insignificant, and there will be no physical basis for the effect.
It's a shame you don't take the scientific method a little more seriously, and approach scientific challenge so defensively.
This, it turns out, wasn't as persuasive as I though it would be. Revere, remarkably, has a soft spot for the discredited idea that low intensity, low frequency, EM fields have physiological effects, and provided a link to a really bad paper claiming some basis for this. That was easily dealt with. But then revere decided to go the
ad hominem route:
Gerard: Yes, that bumblebee can't fly. Everyone is wrong but you. Some scientist. Just disregard the evidence it if doesn't match your preconceived notions. As for ideological bias, what's the name of your blog again? The Right Wing Professor? (as Gerard knows, this is not snark; that's actually the name of his blog). BTW, the way a real scientist would phrase the point he makes is this: "given some numbers I have on binding constants (which estrogen receptor, which model system, under what conditions?) what is the explanation for the observed biological effects?" Instead Gerard says, "I have these numbers and they imply that the biological effects being seen in studies which I haven't bothered to critique but whose reliability I question a priori because they disagree with my libertarian bias not to interfere with the vaunted market can't possibly be true." Some scientist.
I pointed out that Revere hadn't actually provided any evidence. So, to give him his fair dues, he did (while simultaneously accusing me of not knowing there were two types of estrogen receptors, something I'd mentioned previously in the discussion). He linked specificially to two papers. These are
Zsarnovszky A et al. Endocrinol. 2005;146[12]5388-5396 and
Wozniak AL et al. Environ Health Perspect. 2005;113:431-439.
Our crappy librbary doesn't have the first, so I've sent away for it via interlibrary loan. I did, however, find the second paper, as well as a follow up in
Steroids 72:124–134 (2007), and was, frankly, shocked. The investigators applied estrogen (E2), diethylstibestrol, or DES, an authentic synthetic estrogen, as well as several 'xenestrogens', to some prolactin-producting tumor cells, and measured increases in intracellular calcium (an indication of membrane-mdediated signaling) as well as prolactin release. Or so they said in the abstract Their actual data tell an entirely different story.
This is the calcium release data, measured by observing the fluorescence of a dye whose optical properties change when it binds calcium.

Some explanation is needed; the x axis is BPA concentration, and it's in logarithmic units. -12M means 1 picomolar (1 pM = 10--12M), or one trillionth of a mole in 1 Liter of solution. -10 is 100 pM, -9 is 1 nanomolar (1 nM = 1000 pM) and -8M means 10 nM. So, in addition to the untreated cells, they've looked at the concentration over a concentration range spanned by a factor of 10,000. On the y axis, they have the change in fluorescence on adding BPA, as a ratio of the original fluorescence. And it's tiny! Adding 1 pM BPA causes about a 1.2% increase in calcium release. Problem is, add 100 times more, and the very small 1.2% response goes down to 1%!Add 10 times more, and you get an increase to about 4%; add times times more still, and it goes back down to about a 2.5 % increase.
This, my brothers and sisters in science, is what we technically call 'noise'. There is absolutely no physically reasonable response that explains these data as presented. The effects are tiny and show no discernable trend. This becomes abundantly clear if we lose the rather weaselly way the authors presented their data, and simply plot what they measured against what they added, on a linear scale.

The only reasonable conclusion from that graph is that there is absolutely no evidence of any response to BPA, and in fact the data rather suggests there is no response. This is the exact opposite of the conclusion the authors drew.
This is a lengthy post already, and I won't belabor the prolactin release data, except to say there is no time delay in the response, whereas the basic physics of binding very dilute molecules to receptors say there have to be diffusion-limited kinetics (think about it this way: two molecules very far apart have to find each other in order to interact, and that takes time). The concentration graph is equally funky, showing a response at 1 pM and 10 nM, but no response at 10 pM, 100 pM, and 1 nM!
This is genuinely bad science; the data don't support the conclusions, in fact they contradict the conclusions. It's clearly agenda-driven, and it ignores some very basic physics of extremely dilute solutions. Moreover, there is at least one very plausible explanation about why the data are so bad. BPA is, as we said, ubiquitous. Biomedical research labs are full of plastics: they use plastic tubes, plastic pipette tips, tubing, plastic in their water purifiers, etc. Unless you take very stringent steps to eliminate BPA, it is very likely all their solutions have some quite signficant concentration of it. The authors said nothing about this, and so its reasonable to assume they didn't take such steps.
So, after the personal attacks, the defensiveness, etc., this incredibly flawed paper is what revere had to support his point. I think it's more likely than not he never read the paper he cited; he certainly didn't read it with any attention to detail. And that's just bad science, something that's far too prevalent on scienceblogs.com.
Ian Musgrave had the following comments on the above. I'm posting them here in full; I will have my own response to his response later.
Your post on xenoestrogens raised a number of issues; unfortunately, you misunderstand the science in this paper. As I have had a fair bit of experience with measuring intracellular calcium, and have measured intracellular calcium in endocrine cells (eg Ann N Y Acad Sci. 1995 Jul 12;763:272-82. Endocrine. 1998 Aug;9(1):71-7. J Immunol. 2006 Jun 15;176(12):7489-94.), so I would like to comment if I may.
Firstly, endocrine cells such as the GH3 ones used here are a bit of a challenge to measure intracellular calcium ([Ca2+]i) accurately, as they are excitable cells and produce spontaneous oscillations when activated. This necessarily means that the data from activated cells will be somewhat noisy. However, it can quite clearly be seen in the original traces that activation only occurs when drugs (either estrogen, bisphenol A, nonylphenol etc.) are added. Before drug addition, cells are quite stable (see traces in figure 1). Furthermore, when the drugs (including estrogen) are placed on membrane associated estrogen-receptor deficient cells, no activation is seen (figure 3). As the receptor deficient cells still respond to other activators, we can conclude that the effect is specific to membrane bound estrogen receptors. Furthermore, when calcium is omitted from the medium, or in the presence of a calcium channel blocker, the response goes away (eg see figure 6, while the response is not modified by the calcium store releaser thapsigargin). All these results strongly suggest that the effect of BPA (and nonylphenol and endosulfan) are, like estrogen, produced through the membrane associated estrogen receptor.
Part of your critique rests on the apparent size of the response. Firstly, you re-interpret the ratios as percentages. You can't do that as the response is strongly non-linear, a o.o4 increase in fluorescence ratio is not a 4% increase in [Ca2+]i. It has to be interpreted in relation to calibration stimuli such as the 20mM K+ stimuli and the thapsigargin stimulus. In resting GH3 cells, basal [Ca2+]i is roughly 80 nM, the thapsigargin data indicate that estrogen is producing around a 200 nM increase in [Ca2+]i, and a quick back of the envelope calculation for the 20mM K+ stimulus gives roughly the same value. This would mean that the increase produced by BPA is roughly 80-100 nM [Ca2+]i. This is a substantial and physiologically relevant increase.
Another part of the critique rests on the supposed "unphysiological" changes in the graph of BPA. However, biological data is noisy, and we almost never see ideal concentration-response curves in real experiments. The curve for BPA is perfectly compatible with standard biological and experimental variation (and what about nonylphenol, which has pretty much a standard concentration response curve). Also, on the curve, using log values is not "weaselling", it is standard and accepted practice for displaying concentration response curves (in part because of the equations governing receptor occupancy follow log distributions). Pick up any copy of the British Journal of Pharmacology and you will see almost every article using the same approach.
The time course experiments are perfectly ok as well. The smallest time point used was 1 minute (which was the fastest practical under the circumstances), but free diffusion and mixing of ligands with receptors happens on the order of seconds, and the response, especially for excitable cells like GH3 or PC-12 cells (the ones I have worked most with), and indeed be maximal or near maximal within 1 minute (even in slow poke tissues like ileal smooth muscle the response to muscarinic agonists is pretty well maximal by 1 minute)
In summary, the data displays exactly what we would expect if BPA and nonylphenol are acting via estrogen receptors. The increases in fluorescence ratio are consistent with physiological changes in [Ca2+]i, that the "wobblyness" of graphs is with experience of biological and measurement variation. Thus it can be concluded that BPA (and nonylphenol and the other xenoestrogens) act on GH3 cells at low (nanomolar) concentrations that are similar to those of estrogen itself, and act on the membrane bound estrogen alpha receptor via a calcium dependent mechanism.
Cheers! Ian