new stuff from Fox et al.

Fox et al. show that several synthetic compounds that bind with the estrogen receptor—including DDT, bisphenol A and methyl parathion —interfere with the ability of nitrogen-fixing bacteria to form a symbiotic relationship with their leguminaceous hosts (plants like beans, peas and alfalfa). This symbiosis is the basis for a key ecological process, nitrogen fixation, which is essential for life on earth.

Background: Rhizobial bacteria form symbiotic relationships with legumes, living in nodules within the plant's roots and converting nitrogen from one chemical form to another. The conversion, called nitrogen fixation, is the principal natural process by which nitrogen is made available for use by living organisms. Life doesn't work without it.

The plant-bacteria symbiosis is initiated when the bacterium detects a chemical signal exuding naturally from the roots of the legume. The signals belong to a class of compounds, phytoestrogens, which are so described because of their coincidental ability to interact with vertebrate estrogen receptors.

To detect the signal, the bacteria employs receptors analogous to hormone receptors. The phytoestrogen binds to the bacterial receptor and the resulting complex then activates a gene in the bacterium. Activated, the gene initiates an exchange of chemicals between plant and bacteria that stimulates and maintains the nodules in which the bacteria live.

What did they do? Fox et al. reasoned that if phytoestrogens were able to interact with the estrogen receptor, then synthetic compounds that interact with the estrogen receptor might be capable of binding with the bacterium's phytoestrogen receptor and reducing gene activation.

Fox et al. worked with alfalfa and its symbiotic bacterium Sinorhizobium meliloti. The plant exudes a phytoestrogen, the flavenoid, luteolin, which activates the Nod gene in the bacterium.

They created an in vitro testing system in which they could measure Nod gene induction with luteolin alone and then when a series of endocrine disrupting compounds (EDCs) were added to the experiment.

Nod induction by luteolin at 1µ Molar concentration was set as the standard for the experiment, or 100% induction. Adding EDCs separately in different concentrations then allowed Fox et al. to determine the potency of EDCs in suppressing Nod induction.

They performed a second set of experiments with alfalfa roots to determine whether the EDC impact on Nod induction would occur in whole organisms. To do this, they inoculated the roots with a bacterium that turns blue upon exposure to one of the biochemical products of Nod gene activation.

What did they find? Contaminants with estrogenic activity decreased gene expression by up to 90%. In addition to DDT and bisphenol A, methyl parathion, pentachlorophenol and two plant flavonoids (chrysin and genistein ) also interfered with phytoestrogen signaling.

Decreases in Nod induction caused by adding different EDCs to an in vitro assay with 1µM luteolin. In the experiment, the standard for Nod induction (100%) is set by the impact of 1µM luteolin alone. As the concentration of contaminants is increased (from left to right in the graph), Nod induction is reduced. From Fox et al. 2001.

These photographs of alfalfa roots show the impact of several EDCs on Nod gene expression. In the control, the root tip is exposed only to the phytoestrogen luteolin at 1 µM concentration. It is blue over much of its length, reflecting the presence or one of the biochemical products of gene activation. In the lower three photographs, three different compounds are added to the experiment, each at 50 µM. Each decreases the amount of blue, indicating that gene expression has been reduced.   from Fox et al. 2001.

What does this mean?

There are two important lessons from this study.

The first is that it demonstrates conclusively that the symbiosis between legumes and rhizobial bacteria is vulnerable to signal disruption by synthetic contaminants. How extensively this is occuring in the real world becomes an important question, as this symbiosis is crucial to one of the main biogeochemical cycles that makes life on earth possible, the nitrogen cycle. Several of the compounds Fox et al. tested are widespread contaminants in soils. Other contaminants, not tested, share chemical characteristics with those shown in these experiments to have effects, and these others are both likely to interfere with the same process and widely distributed in soils. Thus it is likely that nitrogen fixation has been affected.

The second important lesson from this work is that it reinforces the need to consider endocrine disruption as just one type of chemical impact within a broader framework of signal, or message, disruption. Many of life's crucial processes are controlled by chemical signals. Some of these, hormones, mediate events within and among cells, for example, the activation of specific genes. Fox et al. demonstrate that signal disruption can also take place in chemical message systems controlling relationships between organisms, in this case the two participants in a symbiotic relationship: legumes and rhizobial bacteria.

One of the major trends of endocrine disruption research has been to gradually broaden the range of systems thought to be vulnerable. This field started with a tight focus on contaminants that interfere with estrogen signaling. It broadened to other steroid hormones like testosterone and progesterone, and to non-steroidal hormones like thyroid. Fox et al.'s results take it one major step farther: communication among organisms.

Their results should encourage researchers to begin to look at other chemically-mediated symbioses for signs of chemical disruption. Foremost among these, in my opinion, should be two:

• Coral bleaching threatens coral reefs world-wide. It involves the expulsion of symbiotic algae (zooxanthellae) from their coral hosts. While the bleaching has been widely attributed to increases in sea-water temperature associated with global warming, the changes in temperature that have been experienced are small. An important hypothesis to consider is that the signals mediating this symbiosis have been chemically disrupted, either by chemicals alone or by an interaction between chemicals and rising temperatures. Such interactions would not be unprecedented: for example, temperature and contamination can interact to alter the sex ratio of turtles.
• Scientists have noticed widespread forest decline involving trees in Europe and North America. These declines are at least in part associated with changes in the abundance of mycorrhizal fungi, which exist symbiotically with tree roots and are essential for nutrient absorbtion by tree roots. Investigation into possible disruption of the signals that mediate these symbioses might prove very useful to understand the declines.

New findings from Fox et al.

At the October 2001 ehormone meeting at Tulane University in New Orleans, Jennifer Fox and colleagues presented new data from their studies of the impact of EDCs on symbiosis. In this new set of experiments, they exposed growing plants to EDCs and examined the numbers of nodules formed per plant and the mass of the plants. EDCs suppressed nodule number and plant biomass, as predicted by the study reported in Nature (above). Thus the impact of EDCs on Nod gene activation is likely to have real world effects.

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