Evaluation of the buffer capacity and permeability constant for protons in chromatophores from Rhodobacter capsulatus.

1. The kinetics of decay in the dark of the transmembrane pH difference (delta pH) induced by light in nonphosphorylating chromatophores of Rhodobacter capsulatus were studied using the fluorescent probe 9-aminoacridine, in the presence of 50 mM KCl and 2 microM valinomycin. The transient fluorescence changes induced by acid to base transitions of chromatophore suspensions were used as an empirical calibration [Casadio, R. & Melandri, B. A. (1985) Arch. Biophys. Biochem. 238, 219-228]. The kinetic competence of the probe response was tested by accelerating the delta pH decay with the ionophore nigericin. 2. The time course in the dark of the increase in the internal pH in pre-illuminated chromatophores was analyzed on the basis of a model which assumes a certain number of internal buffers in equilibrium with the free protons and a diffusion-controlled H+ efflux [Whitmarsh, J. (1987) Photosynt. Res. 12, 43-62]. This model was extended to include the effects of the transmembrane electric potential difference on the H+ efflux. 3. The diffusion constant for proton efflux was measured at different values of the internal pH by evaluating the frequency of trains of single-turnover flashes capable of maintaining different delta pH in a steady state. The steady-state equation derived from the model does not include any parameter relative to the internal buffers and allows unequivocal determination of the diffusion constant on the basis of the known H+/e- ratio (equal to two) for the active proton translocation by the bacterial photosynthetic chain. A value for the first-order diffusion constant corresponding to a permeability coefficient, PH = 0.2 micron.s-1, was obtained at an external pH of 8.0; this value was constant for an internal pH ranging over 7.0-4.7. 4. Using the value of the diffusion constant determined experimentally, a satisfactory fitting of the kinetics of delta pH decay in the dark could be obtained when the presence of two internal buffers (with pK values of 3.6 and 6.7, respectively) was assumed. For these calculations, the time course of the transmembrane electric potential difference was evaluated from the electrochromic signal of carotenoids, calibrated with K(+)-induced diffusion potentials. The two internal buffers, suitable for modelling the behaviour of the system, were at concentrations of 250 mM (pK = 3.6) and 24 mM (pK = 6.7) respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzene adducts with rat nucleic acids and proteins: dose-response relationship after treatment in vivo.

The dose-response relationship of the benzene covalent interaction with biological macromolecules from rat organs was studied. The administered dose range was 3.6 x 10(7) starting from the highest dosage employed, 486 mg/kg, which is oncogenic for rodents, and included low and very low dosages. The present study was initially performed with tritium-labeled benzene, administered by IP injection. In order to exclude the possibility that part of the detected radioactivity was due to tritium incorporated into DNA from metabolic processes, 14C-benzene was then also used following a similar experimental design. By HPLC analysis, a single adduct from benzene-treated DNA was detected; adduct identification will be attempted in the near future. Linear dose-response relationship was observed within most of the range of explored doses. Linearity was particularly evident within low and very low dosages. Saturation of benzene metabolism did occur at the highest dosages for most of the assayed macromolecules and organs, especially in rat liver. This finding could be considered as indicative of the dose-response relationship of tumor induction and could be used in risk assessment.

Covalent binding of 1,1,1,2-tetrachloroethane to nucleic acids as evidence of genotoxic activity.

Twenty-two hours after ip administration to male Wistar rats and BALB/c mice, 1,1,1,2-tetrachloroethane (1,1,1,2-TTCE) is bound covalently to DNA, RNA, and proteins of liver, lung, kidney, and stomach. The in vivo reactivity leads to binding values to DNA generally higher in mouse organs than in rat organs. The covalent binding index (CBI) values (82 in mouse liver DNA and 40 in rat liver DNA) classify 1,1,1,2-TTCE as a weak to moderate initiator. Both microsomal and cytosolic enzymatic systems from rat and mouse organs are capable of bioactivating 1,1,1,2-TTCE in vitro. Liver fractions are the most effective. When the activating systems are simultaneously present in the incubation mixture a synergistic effect is observed. Unlike the related chemical 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE), which is bioactivated only through an oxidative route, 1,1,1,2-TTCE metabolism is carried on by oxidative and reductive pathways, both dependent on cytochrome P-450. 1,1,1,2-TTCE is also bioactivated by microsomal GSH-transferases from liver and lung. These data further confirm that correlations exist between structure and genotoxic activity of halocompounds.

Metabolic activation and covalent binding to nucleic acids of pentachloroethane as short-term test of genotoxicity.

The in vivo covalent binding of 14C-pentachloroethane to DNA, RNA and proteins of rats and mouse organs was detected 22 hr after i.p. injection. The covalent binding index, calculated on the liver labeling was comparable to those of compounds considered as weak-moderate initiators. Like other haloalkanes, 14C-pentachloroethane was bioactivated in in vitro cell-free system by both microsomal and cytosolic enzymatic fractions from mouse and rat organs to react covalently with DNA and other macromolecules. The binding extents obtained from in vitro incubation and the binding values detected after in vivo administration of labeled pentachloroethane were comparable each other and showed a high correlation with oncogenic potency index of this compound. This result confirms the efficiency of in vitro binding as short-term test of genotoxicity prediction.

Binding of hexachloroethane to biological macromolecules from rat and mouse organs.

Hexachloroethane (HCE) binds to macromolecules of rat and mouse both in vivo and in vitro after metabolic activation. The covalent binding index (CBI) to liver DNA in vivo is comparable to that of compounds classified as weak-moderate initiators and is of approximately the same order of magnitude as those of other halocompounds such as 1,2-dichloroethane. HCE is bioactivated in vitro by microsomal enzymatic systems from murine liver and kidney and, to a greater extent, by cytosolic fractions from all assayed organs. HCE is less reactive than 1,1,2,2-tetrachloroethane, which is more toxic and oncogenic. The ability of hexachloroethane and five other chloroethanes to react covalently with mouse liver DNA both in vivo and in vitro parallels the relative oncogenic potency of these hepatocarcinogenic chemicals in mouse liver.

Evidence of DNA binding activity of perchloroethylene.

14C-Perchloroethylene is covalently bound to DNA, RNA and proteins of rat and mouse organs in vivo after ip injection. Covalent Binding Index values are typical of weak-moderate and weak initiators, for mouse and rat liver, respectively. The greater amounts of labelings detected in mouse liver and in rat kidney macromolecules are consistent with the known toxic and carcinogenic actions of this compound. In vitro binding of perchloroethylene to nucleic acids and proteins proceeds through the involvement of the P-450-dependent mixed function oxidase system from liver microsomes. Kidney, lung and stomach microsomal fractions are uneffective. Cytosolic enzymes from all assayed organs are much more efficient than liver microsomes in bioactivating the compound. GSH addition to liver microsomal system greatly enhances binding extent. This observation suggests that GSH plays a role in the binding of perchloroethylene metabolites as for symmetrically substituted haloethanes.

Short-term tests of genotoxicity for 1,1,1-trichloroethane.

Covalent binding of 14C-1,1,1-trichloroethane to macromolecules from rat and mouse liver, kidney, lung and stomach was analyzed under the same experimental conditions previously utilized in studying 1,1-dichloroethane and 1,1,2-trichloroethane. Labeling of DNA, RNA and proteins was very low both in in vivo interaction and in in vitro microsome-mediated binding. Interaction proceeded through the involvement of the P-450-dependent mixed function oxidase system from liver microsomes and, to a lesser extent, from lung microsomes. Covalent Binding Index of 1,1,1-trichloroethane in liver DNA was typical of very weak initiators. However, overall evaluation of the short-term assays available for 1,1,1-trichloroethane leads to limited evidence of genotoxicity. On the other hand, the evidence of 1,1,1-trichloroethane carcinogenicity in animals is still inadequate.