Biomarkers of drug-induced vascular injury.

In pre-clinical safety studies, drug-induced vascular injury is an issue of concern because there are no obvious diagnostic markers for pre-clinical or clinical monitoring and there is an intellectual gap in our understanding of the pathogenesis of this lesion. While vasodilatation and increased shear stress appear to play a role, the exact mechanism(s) of injury to the primary targets, smooth muscle and endothelial cells are unknown. However, evaluation of novel markers for potential clinical monitoring with a mechanistic underpinning would add value in risk assessment and management. This mini review focuses on the progress to identify diagnostic markers of drug-induced vascular injury. Von Willebrand factor (vWF), released upon perturbation of endothelial cells, is transiently increased in plasma prior to morphological evidence of damage in dogs or rats treated with vascular toxicants. Therefore, vWF might be a predictive biomarker of vascular injury. However, vWF is not an appropriate biomarker of lesion progression or severity since levels return to baseline values when there is morphological evidence of injury. A potential mechanistically linked biomarker of vascular injury is caveolin-1. Expression of this protein, localized primarily to smooth muscle and endothelial cells, decreases with the onset of vascular damage. Since vascular injury involves multiple mediators and cell types, evaluation of a panel rather than a single biomarker may be more useful in monitoring early and severe progressive vascular injury.

Inhibition of cell-cell communication by methylsulfonyl metabolites of polychlorinated biphenyl congeners in rat liver epithelial IAR 20 cells.

The effects of three polychlorinated biphenyl (PCB) congeners and their six methylsulfonyl (MeSO2)-metabolites on cell communication have been investigated in the scrape-loading/dye-transfer assay in IAR 20 rat liver epithelial cells. The results demonstrated that at non-cytotoxic concentrations 2,2',4',5-tetrachlorobiphenyl, 2,2',4',5,5'-pentachlorobiphenyl (2,2',4',5,5'-pentaCB), 2,2',4',5,5',6-hexachlorobiphenyl (2,2',4',5,5', 6-hexaCB), and their 3- and 4-MeSO2 derivatives completely inhibited the cell communication within 1 h. 4-MeSO2-2,2',4',5,5'-pentaCB and 4-MeSO2-2,2',4',5, 5',6-hexaCB appeared to inhibit the cell communication at slightly lower concentration than their parental PCB congeners and 3-MeSO2 derivatives. The results show that 3- and 4-MeSO2 derivatives of the PCB congeners tested inhibit gap junction intercellular communication at about the same potency as their parental compounds. Since inhibition of cell communication is often observed after treatment with many tumor promoters, our findings suggest that the metabolites may also act as tumor promoters.

The ability to alter the gap junction protein expression outside GST-P positive foci in liver of rats was associated to the tumour promotion potency of different polychlorinated biphenyls.

The results demonstrate different modes of action by a dioxin-like polychlorinated biphenyl (PCB 126) and a non dioxin-like PCB (PCB 153) in the alteration of connexin (cx) 26 and cx 32 expression outside GST-P positive foci in liver of female Sprague-Dawley rats, treated according to an initiation-promotion protocol. A decreased relative amount of immunopositive cx 26 and cx 32 spots in the parenchymal cell plasma membranes was observed after treatment with the potent tumour promoters PCB 126 or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). No reduction of cx 26 or cx 32 was noted after administration with the weaker tumour promoters PCB 153 or PCB 118 (PCB 118; both dioxin- and non dioxin-like). Additionally, we found that the down-regulation of connexins also occurred in rats treated with PCB 126 or TCDD without partial hepatectomy and initiation with nitrosodiethylamine. In summary, the results indicate that the ability to reduce the gap junction protein level in liver of rats can be associated to the tumour promotive potency of the different PCB-congeners and TCDD.

Inhibition of cell-cell communication by commercial chlorinated paraffins in rat liver epithelial IAR 20 cells.

The effects of six commercial chlorinated paraffins of different carbon chains length and chlorine content (Cereclor 50LV [C50LV], Hüls 60 [H60], Cereclor S45 [CS45], Cereclor S52 [CS52], Cereclor 42 [C42] and Cereclor 48 [C48] on cell communication have been investigated in the scrape-loading/dye-transfer assay in IAR 20 rat liver epithelial cells, as well as the effects of these compounds on connexin 43 (cx 43), the main gap junction protein in this cell line. The results clearly demonstrated that at non-cytotoxic concentrations C50LV, H60, CS45 and CS52 completely inhibited the cell communication within 1 hr. The short carbon chain length chlorinated paraffins (C50LV and H60) were inhibiting the cell communication at lower concentration than the intermediate carbon chain length chlorinated paraffins (CS45 and CS52). Almost complete inhibition of the cell communication was maintained for at least 24 hrs of H60 exposure. Immunoblots of IAR 20 cell extracts after H60-exposure showed a decreased phosphorylation of cx 43 after 1, 4 and 24 hrs of treatment. The phosphorylation pattern of cx 43 prepared from H60- or CS52-exposed cells was different from that prepared from 12-O-tetradecanoylphobol-13-acetate (TPA)-exposed cells after 1 hr treatment. The results show that the short and intermediate, but not the long carbon chain length chlorinated paraffins, are potent inhibitors of gap junction intercellular communication. Thus, our findings suggest that these compounds may act as tumour promoters.

Mechanistical studies of the inhibition of intercellular communication by organochlorine compounds.

Many hydrocarbons are environmental pollutants that, due to their lipophilicity and chemical stability, accumulate in biological systems including milk and body fat. A number of investigations have demonstrated that many organochlorine compounds can act as tumour promoters in vivo and inhibit gap junctional intercellular communication between cells in culture. In the present study we have investigated the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), different polychlorinated biphenyls, chlorinated paraffins and the pesticide endosulfan. Using techniques of scrape loading dye/transfer and Western blot analysis the function, expression and phosphorylation of different connexins in vitro and in vivo were studied. The results show a good correlation between the ability to act as a tumour promoter and to interfere with gap junctional intercellular communication. All tested compounds inhibited the intercellular communication in a liver derived cell line (IAR 20). However, the results show that the time to inhibition varies between the different agents. Endosulfan and chlorinated paraffins inhibit the communication within one hour, whereas dioxin like substances need to expose the cells for 48 hours before the communication is affected.

The human RAD51/RecA homologue gene is not a candidate gene for Bloom's syndrome.

The human gene HSRAD51/RecA homologue has been investigated as a possible candidate gene involved in Bloom's syndrome. No mutations were found in the cDNA isolated from three different Bloom's syndrome cell lines, thus excluding the possibility that HSRAD51 is directly involved in the syndrome. Other possible candidates are discussed.

Alteration in expression of gap junction proteins in rat liver after treatment with the tumour promoter 3,4,5,3',4'-pentachlorobiphenyl.

Polychlorinated biphenyls (PCBs) are industrial chemicals which are highly persistent and widely distributed in the environment. We have previously shown that 3,4,5,3',4'-pentachlorobiphenyl (PCB 126) is a potent tumour promoter in two separate 20 week initiation-promotion studies. In the present study, rat livers from these two studies were further investigated for connexin expression. The results demonstrated that treatment with PCB 126 caused a decrease in the amount of the two major liver connexins, cx 26 and cx 32, in livers of treated animals. This reduction was also prominent after treatment at low doses, although gamma-glutamyl transpeptidase-positive foci had not developed in these livers. The quantity of cx 26 and cx 32 in immunostained liver sections was determined using a computerized fluorescence image analyzer. Western blot analysis of liver extracts confirmed these results. No changes in the RNA levels in the treated rats were seen, suggesting that the down-regulation of cx 26 and cx 32 is post-transcriptional.

Two inhibitors of gap junctional intercellular communication, TPA and endosulfan: different effects on phosphorylation of connexin 43 in the rat liver epithelial cell line, IAR 20.

The skin tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the chlorinated insecticide, endosulfan, are two potent inhibitors of gap junctional intercellular communication. In the present study the effects of TPA and endosulfan on cell communication have been investigated in IAR 20 rat liver epithelial cells, as well as the effects of these compounds on connexin 43 (cx43), the main gap junction protein in this cell line. The results clearly demonstrate that at non-toxic doses both compounds inhibited the cell communication by at least 90% within 5 min. The communication was partially restored after 4 h of TPA exposure and almost fully restored by 24 h, whereas in endosulfan-exposed cells the communication was completely down-regulated for the whole exposure-period of 24 h. Immunoblots of IAR 20 cell extracts indicated that TPA initially caused an increased phosphorylation of cx43. A normal phosphorylation pattern was observed after 4 h when the cell communication was restored. Immunoblot analysis after endosulfan-exposure showed a slightly increased phosphorylation of cx43 after 10 min treatment, gradually followed by dephosphorylation during the rest of the 24 h treatment period. Immunostaining of IAR 20 cells showed that both compounds caused a rapid disappearance of cx43 from the cell membrane. After 4 h of exposure immunofluorescent cx43-plaques started to reappear in the cell membrane, although less pronounced in endosulfan-treated cells. However, after 24 h of endosulfan-exposure a high number of cx43-spots was demonstrated. These results indicate that different mechanisms are responsible for the inhibition of gap junctional intercellular communication induced by TPA and by endosulfan, at least during the later part of the 24 h exposure-period. TPA causes a marked hyperphosphorylation of cx43, whereas endosulfan increases phosphorylation initially only slightly but longer exposure-periods lead to hypophosphorylation. Thus, phosphorylation as well as dephosphorylation seem to be important factors involved in the regulation of the function of cx43 in this cell line.

Altered DNA ligase III activity in the CHO EM9 mutant.

Delayed joining of DNA strand breaks and a high spontaneous level of sister-chromatid exchanges (SCEs) are characteristics of the mutant cell strain EM9 of Chinese hamster ovary (CHO) cells. The introduction of the human gene XRCC1 into EM9 cells reverts the phenotypic properties of EM9 to those of the wild type. We have investigated both DNA ligase activities and a protein which stimulates DNA ligase activity in mutant EM9 cells, XRCC1-transfectant H9T3-7-1 cells and wild-type AA8 cells. Our results, which demonstrate both a decreased DNA ligase activity in EM9 cells using poly(rA).oligo(dT) as substrate and a decreased ability of DNA ligase III to form a covalent DNA ligase III-adenylate intermediate with AMP, clearly indicate an altered DNA ligase III activity in the mutant. Furthermore, the AMP-binding capacity of DNA ligase III and its enzymatic activity with the synthetic polymer were restored after transfection of EM9 with the human XRCC1 gene. Immunoblotting data suggest that the XRCC1 gene does not code for DNA ligase III. In conclusion, the data indicate that the EM9 cell strain has an altered DNA ligase III activity that can be restored by the XRCC1 gene product.

Deoxyribonucleoside triphosphate pool levels in three cell strains of human chromosome instability syndromes: ataxia telangiectasia (GM2052), Bloom's syndrome (GM1492), and Fanconi's anemia (GM368).

Deoxyribonucleoside triphosphate (dNTP) pool sizes were determined in cell strains derived from patients with the genetic diseases ataxia telangiectasia (GM2052), Bloom's syndrome (GM1492), and Fanconi's anemia (GM368), and were compared to the dNTP pools in a normal human fibroblast cell strain (253/79). In addition, the effect of deoxythymidine on both dNTP pool levels and cell growth was examined. The three mutant cell strains differed only slightly from the normal cell strain. The cellular characteristics of the cell strains, such as chromosome instability, are apparently not an effect of dNTP pool imbalance.

Expression of a DNA-ligase-stimulatory factor in Bloom's syndrome cell line GM1492.

An increased DNA ligase activity is observed in extracts of Bloom's syndrome (BS) fibroblast cell line GM1492. The activity is 2-3-fold higher in this cell line compared to normal human fibroblasts, and 5-20-fold higher than in three other BS cell lines investigated. The DNA ligase activity in GM1492 cells is promoted by a heat-resistant, protease-sensitive factor comigrating with DNA ligases on single-stranded-DNA--cellulose. The factor stimulates DNA ligase I as well as DNA ligase II, and is not identical to the activity-promoting homologous DNA pairing, which is also enhanced in GM1492 cell extracts.

RecA-like activity in mammalian cell extracts of different origin.

The occurrence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Bloom's syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from phi X174 RFI DNA and fragments of phi X174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4-7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Bloom's syndrome fibroblast cell lines, Fanconi's anemia fibroblasts, virus- (Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.

Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese hamster ovary cells.

The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo[a]pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 microns), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possibly be used as an index of the degree of mitotic disturbances induced by mineral fibers.

A DNA-recombinogenic activity in human cells.

A DNA recombining protein has been partly purified from cell lines derived from patients suffering from the hereditary disease, Bloom's syndrome. The protein induces the formation of displacement loops in phi X174 RFI DNA molecules after the addition of single-stranded DNA fragments. A filter binding method and electron microscopy were used to determine the reaction. The recombinogenic protein is dependent on divalent cations and ATP for activity.

Insect pathogenic properties of Serratia marcescens: phage-resistant mutants with a decreased resistance to Cecropia immunity and a decreased virulence to Drosophila.

A non-pigmented strain of Serratia marcescens (Db10) was isolated from moribund Drosophila flies. From this strain were isolated spontaneous mutants resistant to streptomycin (Db11) and nalidixic acid (Db12). Mutant Db11 was used for the isolation of two phages, phi J and phi K, which grew on Db10, Db11 and Db12, but not on three reference strains of S. marcescens. Mutant Db11 was demonstrated to fulfil koch's postulates. Strain Db10 and its antibiotic-resistant derivatives were lethal to Drosophila whether given in the food or by injection. Evidence for toxin(s) was found only in sterile supernatants from 7 d cultures. Such extracts contained proteolytic activity and inactivated the antibacterial activity in immune haemolymph from Cecropia. Phages phi J and phi K were used to isolate phage-resistant mutants of Db11. Three such mutants and their parental strain were investigated for their susceptibility to immune haemolymph from Cecropia. The parental strain was resistant to incubation with 90% haemolymph for 2 h at 37 degrees C; all phage-resistant mutants were susceptible to the immune haemolymph with "killing times" (i.e. the time required to kill 90% of the viable cells) ranging from 15 to 55 min. When the same strains were compared for their virulence to Drosophila, the phage-resistant mutants had significantly reduced virulence. It is concluded that resistance to insect immunity plays an important role in the overall pathogenicity of S. marcescens.