UV-induced photolesions, their repair and mutations.

UV-induced cyclobutane pyrimidine dimers (CPD) are selectively removed from the transcribed strand of transcriptionally active genes in V79 Chinese hamster cells. This strand specificity of repair corresponds well with the observation that UV-induced mutations in the HPRT gene are primarily generated by DNA photolesions in the non-transcribed strand. This strand bias for mutations is, however, much more pronounced at 2 J/m2 than at the higher dose of 12 J/m2. An alternative explanation for strand specificity of mutations would be that most of the mutations are caused by pyrimidone 6-4 pyrimidine photoproducts (6-4 PP). Indeed experiments with a V79-derived cell line capable of repairing 6-4 PP but not CPD have revealed direct evidence for 6-4 PP as the mutagenic lesions in UV-irradiated hamster cells. This implies that 6-4 PP are also preferentially repaired in the transcribed strand. We have investigated the repair of DNA photolesions in the HPRT gene by measuring the distribution of bromodeoxyuridine-labeled repair patches in the transcribed and non-transcribed strands of genes employing a newly developed immunoextraction procedure. Three cell lines with different capacities to remove CPD and 6-4 PP from the HPRT gene and from the genome overall were used. We found no evidence for preferential repair of 6-4 PP in the transcribed strand of the HPRT gene in cells exposed to 10 J/m2. These data are in favor of a lack of strand-specific repair of 6-4 PP underlying the much less pronounced strand bias for induced mutations at high UV dose. However, the conclusive test would be the demonstration of preferential repair of 6-4 PP in the transcribed strand of transcriptionally active genes in cells exposed to 2 J/m2.

The use of streptavidin-coated magnetic beads and biotinylated antibodies to investigate induction and repair of DNA damage: analysis of repair patches in specific sequences of uv-irradiated human fibroblasts.

A new immunoextraction method using biotinylated antibodies and streptavidin-coated magnetic beads has been developed and applied to study the repair of uv-induced DNA damage in specific DNA sequences. uv-irradiated cells were allowed to carry out DNA repair for various time intervals in the presence of 5-bromodeoxyuridine (BrdU). Purified and restricted DNA was subjected to an immunoextraction method employing an anti-BrdU antibody (alpha Brdu), biotinylated goat antimouse antibodies (G alpha Mbio), and streptavidin-coated polymeric magnetic beads. Separation of BrdU containing DNA was achieved by using a magnetic device. This extraction procedure resulted in two fractions of DNA, i.e., BrdU-containing and non-BrdU-containing DNA. Both fractions were blotted on filters and subsequently hybridized with specific DNA probes to determine the relative amount of defined fragments in the two fractions of DNA. Repair experiments using normal primary human fibroblasts showed no difference in the incorporation of repair label in the active adenosine deaminase gene and the inactive 754 locus during the first 4 h following uv irradiation. After longer repair times the active gene incorporated more repair label than the inactive gene, consistent with the known preferential repair of cyclobutane pyrimidine dimers from active housekeeping genes.

Comparative study of the effects of four cephalosporins against Escherichia coli in vitro and in vivo.

A thigh muscle infection induced with Escherichia coli in irradiated mice was used as a model to compare the in vivo pharmacodynamics of the antibacterial effect of four cephalosporins (i.e., cefepime, ceftriaxone, ceftazidime, and cefoperazone) with the in vitro antibacterial pharmacodynamics of these drugs. The following in vitro pharmacodynamic parameters were determined: the maximum effect as a measure for efficacy, the 50% effective concentration as a parameter for potency, and the slope of the concentration-effect relationship. For analysis of the in vivo antibacterial pharmacodynamics, the same parameters were applied for the dose instead of the concentration. For the detection of a relationship between concentration and antibacterial effect in vivo, we determined the pharmacokinetics of the four cephalosporins in the plasma of mice. The results showed that, in general, there is a direct relationship between the in vivo and in vitro pharmacodynamics of these cephalosporins. The maximum effects of cefepime, ceftazidime, and cefoperazone were approximately similar in vivo and in vitro. The sequence of potency of these drugs was, in descending order, cefepime, ceftazidime, and cefoperazone. Ceftriaxone differed from the other three cephalosporins in that it displayed unexpected in vivo pharmacodynamics. Ceftriaxone was just as efficacious as the other three in vitro, but its maximum effect in vivo was much lower. This relatively low maximum effect of ceftriaxone in vivo was not explained by the pharmacokinetic characteristics of the drug. From the present results it can be concluded that the in vitro efficacy of cephalosporins does not necessarily have a predictive value for the in vivo efficacy.

Antistaphylococcal activities of teicoplanin and vancomycin in vitro and in an experimental infection.

The efficacies of vancomycin and teicoplanin in an experimental Staphylococcus aureus infection in granulocytopenic mice were related to their activities in vitro and their pharmacokinetic profiles. In vitro teicoplanin had a higher intrinsic activity than vancomycin did; and it also had a more favorable pharmacokinetic profile, resulting in higher peak concentrations in plasma, a longer elimination half-life, and a larger area under the concentration-time curve than those of vancomycin. To predict the antibacterial efficacies of the drugs in vivo on the basis of their activities in vitro and pharmacokinetics, a mathematical model was applied. In the model the in vitro effect was expressed as the difference in growth rate between control cultures and those in the presence of the antibiotic (ER), and the in vivo effect was expressed as the difference between numbers of CFU in control and antibiotic-treated animals (EN). The integral of ER against time, ERt, was calculated by using the concentrations found in vivo. A significant linear relationship was found between EN and ERt for different dosages at the same times (4 h) after drug administration as well as for the same doses at consecutive times, although at the lowest doses of teicoplanin the observed effect was less than the predicted effect.

Antibacterial activity of four cephalosporins in an experimental infection in relation to in vitro effect and pharmacokinetics.

The in vitro activity of four cephalosporins was compared with their effects in an experimental thigh infection (cefuroxime and cefamandole against Escherichia coli and cefamandole, ceftriaxone, and ceftazidime against Klebsiella pneumoniae) in granulocytopenic mice. The effect in vitro (ER) was defined as the difference between the growth rate without antibiotic and the growth rate at the steepest part of a 3-h growth curve in the presence of an antibiotic. The relation between concentration and ER was described with the Hill equation. Using pharmacokinetic parameters of the plasma concentrations in vivo and those of the Hill equation the corresponding time course of ER was calculated and by integration with respect to time (0tERdt), an estimate was obtained of the effect on bacteria. For all four antibiotics this estimate was significantly correlated with the actual values of the effect in vivo (EN), defined as the difference in numbers of bacteria between controls and antibiotic-treated animals at 4 h.