The effects of organic pesticides on inner membrane permeability in Escherichia coli ML35

We have tested whether some pesticides might cause inner membrane leakage in ML35 Escherichia coli cells, which express beta-galactosidase (lacZ; EC 3.2.1.23) constitutively but lack the permease (lacY) required for substrate entry. The activity of beta-galactosidase (indicative of substrate leakage through the inner membrane) was increased by various concentrations of pesticides, including the organometallic fungicides maneb and mancozeb, the insecticide Thiodan, and the herbicide Ally, as well as by antibiotics such as ampicillin, gramicidin D, and the calcium ionophore A23187. The enzyme activity was increased by up to approximately 30% when the E. coli ML35 strain was exposed to various concentrations (between 50 and 250 ppm) of both fungicides. Thiodan had only a slight effect on beta-galactosidase activity (increase of 12.8%), whereas, among the antibiotics, the calcium ionophore at 20 microg/ml caused a significant increase in enzyme activity by up to 61.8%. This effect is similar to that of sodium dodecyl sulfate, used as positive control ( approximately 70% increase). Accumulation of maneb and mancozeb by bacterial cells was also studied taking advantage of their metal content and using atomic absorption spectrophotometry. In parallel with the increase in enzyme activity, both fungicides accumulated in the cells as a function of their concentration. Time course experiments (3, 6, and 9 h) of fungicide accumulation and of bacterial growth at various pesticide concentrations were also carried out. Maneb seems to inhibit the bacterial growth better than mancozeb. In addition, maneb uptake increases with time up to 9 h at all tested concentrations, whereas the accumulation of mancozeb is similar at all the exposure times tested. This indicates a different uptake and/or metabolizing strategy by E. coli cells for the two fungicides.