Nonlinear changes in the activity of the oxygen-dependent demethylase system in Rhodococcus erythropolis cells in the presence of low and very low doses of formaldehyde

Abstract

The effect of exogenous, highly diluted formaldehyde on the rate of demethylation/re-methylation of veratric acid by the bacteria Rhodococcus erythropolis was studied using electrophoretic and microscopic techniques. The activity of 4-O-demethylase, responsible for accumulation of vanillic acid, and the levels of veratric and vanillic acids were determined using capillary electrophoresis. Formaldehyde was serially diluted at 1:100 ratios, and the total number of iterations was 20. After incubation of the successive dilutions of formaldehyde with the bacteria, demethylase activity oscillated in a sinusoidal manner. It was established using capillary electrophoresis that methylation of vanillic acid to veratric acid occurred at a double rate, as shown by the doubled fluctuation in the concentration of veratrate. There were also changes in the NADH oxidase activity, which is associated with methylation processes. Microscopic observations revealed the presence of numerous enlarged vacuoles in bacterial cells during the accumulation of large amounts of vanillic acid, and their disappearance together with a decrease in 4-O-demethylase activity. The presented results give evidence for the ability of living cells to detect the presence of submolecular concentrations of biological effectors in their environment and provide a basis for a scientific explanation of the law of hormesis and the therapeutic effect of homeopathic dilutions.

Figure 1

The hypothetical mechanism of veratrate demethylation to vanillate by Rhodococcus erythropolis cells, dependent on activated state of formaldehyde (Malarczyk and Pazdzioch-Czochra, 2000) [17]. Reproduced with permission of the Biochemical Society. Modified. Abbreviations: HCHO and H*CHO-formaldehyde in normal and activated state; 4-O-D-4-O-Demethylase system, Ox.NADH-NADH oxidase system, MAX state-maximal oxygen uptake, MIN state-maximal oxygen release. More details in the text of the original paper.

Figure 2

The demethylation of veratric acid by Rhodococcus erythropolis cells after 6-hour incubation with successive FA dilutions in water or in 75% ethanol, together with control values for water and 75% ethanol.

Figure 3

Demethylation of veratric acid by Rhodococcus erythropolis cells in the presence of low doses of HCHO as a function of time. Planar version (2D).

Figure 4

Demethylation of veratric acid by Rhodococcus erythropolis cells in the presence of low doses of HCHO as a function in time. Spatial version (3D).

Figure 5

Chromatogram (MEKC) of veratric, vanillic, isovanillic acid and catechol standards.

Figure 6

Changes in the level of veratric acid during incubation of formaldehyde with Rh. erythropolis cells in a series of HCHO dilutions in ethanol and the corresponding levels of vanillic acid identified using MEKC.

Figure 7

Changes in the level of veratric acid during incubation of formaldehyde with Rh. erythropolis cells in a series of HCHO dilutions in water and the corresponding levels of vanillic acid identified using MEKC.

Figure 8

A comparison between demethylation of Rh. erythropolis cells and their oxygenase activity against NADH. Ethanol series.

Figure 9

A comparison between demethylation of Rh. erythropolis cells and their oxygenase activity against NADH. Water series.

Figure 10

Differences in morphology of Rh. erythropolis cells under the action of very low doses of formaldehyde in 75% ethanol. A-control with ethanol; B and C-maximum of demethylation, n = 5 and 15; D and E-minimum of demethylation, n = 10 and 20.