W. Marek

Increased Incidence of DNA Double-strand Breaks and Anti-ds DNA Antibodies in Blood of Workers Occupationally Exposed to Asbestos

Asbestos, proven to be carcinogenic in humans and animals, is reported to have no genotoxic effect. Asbestos workers have an increased risk of lung cancer, mesothelioma, and other tumours. Earlier findings showed that crocidolite can induce DNA strand breaks in cultured rat embryo cells as assessed by nick translation. We investigated DNA double-strand breaks in white blood cells (WBC) of ten workers occupationally exposed to asbestos. According to our results, obtained with neutral filter elution, individuals who had been exposed to asbestos fibres showed two to four times more DNA double-strand breaks (dsb) in white blood cells than ten non-exposed persons. The induced DNA fragments are of about 250 kb (compared to chromosomal DNA of Saccharomyces cerevisiae standard marker). Using additionally the chromosomal DNA protective method of agarose-plugs, DNA fragments in the range of 200 to 1000 kb have been found in the white blood cells of the same ten workers occupationally exposed to asbestos. In the white blood cells of non-exposed subjects no DNA fragments could be detected with this method. Compared to 51 non-exposed persons, elevated anti-ds DNA antibody concentrations were found in ten workers occupationally exposed to asbestos. The fact that workers occupationally exposed to asbestos have distinctly more double-strand breaks and anti-ds DNA antibodies could mean that an increased incidence of DNA-fragments may be an important indicator in the chronic effect of asbestos-associated carcinogenesis. Apparently, the chronic effects of asbestos observed here do not seem to be identical with that of previously reported acute in vitro effects.

DNA damage in human white blood cells after inhalative exposure to methylenediphenyl diisocyanate (MDI) — case report

A workplace-related inhalative exposure test in a challenge chamber was performed on an industrial worker in a methylenediphenyl diisocyanate (MDI) atmosphere from 5 to 20 ppb and the isolated white blood cell DNA was analysed by electrophoresis, anion-exchange chromatography and melting behaviour. The results of electrophoresis indicate that inhaled MDI induces double-strand breaks of DNA. Some of the DNA fragments were estimated to be in the region of 100-500 bp. Anion-exchange chromatography confirmed this finding. Following denaturation and rapid renaturation the results demonstrated that some DNA fragments are cross-linked by MDI. Comparing the melting curves before and after inhalative exposure in the challenge test chamber, genomic DNA revealed differences in the shape of the melting curve (hyperchromic effect). The results suggest that occupational MDI exposure could be associated with white blood cell DNA damage.

INCREASED INCIDENCE OF DNA DOUBLE-STRAND BREAKS IN LUNG AND LIVER OF RATS AFTER EXPOSURE TO CROCIDOLITE ASBESTOS FIBERS

AbstractThe aim of this study was to find out whether UICC crocidolite asbestos fibers induce in vivo DNA double-strand breaks in the nuclei of cells in lung and liver of rats. The effect of crocidolite on chromosomal DNA of rats exposed to crocidolite fibers was analyzed by pulsed-field gel electrophoresis (PFGE) using the chromosomal DNA-protective agarose-plugs method and laser densitometric evaluation of stained DNA bands. The profiles of laser densitometric scanning showing PFGE separation of DNA for exposed rats are right-shifted in liver and lung compared with the controls of the same age. DNA from the liver of nonexposed rats showed one peak in the range of 250–850 kb. Compared to this finding, at 4, 13, and 16 mo after crocidolite instillation DNA of rats showed shorter DNA fragments in the range of 250–700 kb. This indicates a more intact DNA in controls. At 4, 13, and 16 mo after crocidolite instillation, we also found DNA fragments in exposed lungs in the region between 250 and 2200 kb produce...

Indication of Dna Strand Breaks in Human White Blood Cells after In Vitro Exposure to Toluene Diisocyanate (TDI)

Toluene diisocyanate (TDI), used expecially for the production of polyurethanes, is known to induce chromosome aberrations, base-pair substitution, and frameshift mutation after metabolic activation. Following treatment of human blood by TDI, the isolated DNA was analyzed by anion-exchange chromatography (FPLC) before and after denaturation. In addition, DNA from white blood cells was analyzed by alkaline and neutral filter-elution and pulsed-field gel electrophoresis (PFGE). The results show that TDI induced single-and double-strand breaks in the DNA of white blood cells in vitro. The elution rate, calculated after alkaline filter elution, was significantly increased after TDI treatment. An average size of the TDI-induced DNA fragments was estimated by PFGE to be smaller than 250 kb. Denaturation and renaturation of TDI-treated DNA indicated that DNA could be cross-linked by TDI. Purified DNA treated with TDI in buffer alone does not induce DNA fragments as shown by FPLC. These findings indicate that DNA damages are induced by TDI after the biotransformation of TDI. The results show that TDI exposure induces DNA damage of white blood cells in vitro.

Cellular and mediator profile in bronchoalveolar lavage of guinea pigs after toluene diisocyanate (TDI) exposure.

Toluene diisocyanate (TDI) is a volatile, highly reactive chemical widely used as a polymerizing agent in the production of polyurethane foams, lacquers, adhesives, and other items. Repeated airway exposures in the workplace to TDI may cause a concentration-dependent risk of developing chronic airway disorders. Different pathomechanisms are involved. IgE-mediated sensitization and irritative effects were clearly demonstrated in exposed subjects as well as in animals. In this study we examined the cellular and mediator composition in bronchoalveolar lavage fluid (BALF) of guinea pigs (eight in each group) exposed to TDI (10, 20, or 30 ppb) on 5 consecutive days for 2 hours each. Increased numbers of eosinophils and significantly elevated levels of LTB4 and LTC4/LTD4/LTE4 were obtained in BALF of all exposed animals when compared to nonexposed control animals. PGD2 and TXB2 remained unaltered in BALF. Stimulation of BALF cells of exposed and control animals with Ca-ionophore A23187 and arachidonic acid induced an increased generation of LTB4. Furthermore, BALF cells of the exposed animal groups generated immunoreactive LTC4/LTD4/LTE4, whereas controls did not show peptido-leukotriene formation in the presence and absence of stimuli. Our data clearly demonstrate an influx of eosinophils into the airways associated with mediator release and higher cellular responsiveness after TDI exposure.

Hexamethylene Diisocyanate Induction of Transient Airway Hyperresponsiveness in Guinea Pigs

The induction of lung injury and the development of airway hyperresponsiveness (AHR) by exposure to hexamethylene diisocyanate (HDI) were studied in a guinea pig model of occupational lung diseases. In addition to an unexposed control group of 16 guinea pigs (A), two groups (B, C) of 8 animals inhaled HDI atmospheres in the range of the threshold limit value (TLV) of 10 ppb for 6 h/day on 5 days/week over a period of 8 weeks. Airway responses to aerosols of 0.125, 0.25, 0.5, 1.0 and 2.0% acetylcholine (ACH) were measured in exposed as well as in unexposed animals. Basal values of respiratory mechanical and cardiovascular parameters were not significantly altered after 8 weeks of HDI inhalation (group B). Furthermore, additional acute challenge by 10 ppb HDI for a period of 60 min, performed under continuous registration of respiratory and cardiovascular parameters, did not cause any significant changes in functional parameters. After 8 weeks of HDI exposure, the amplitude of airway constriction as a response to 2.0% ACH, indicated by the changes in dynamic elastance (Edyn) rose significantly to almost 5 times the ACH response in group A(p < 0.0005). In group C of 8 guinea pigs, ACH response was evaluated after a latency period of 8 weeks. In this group, changes of airway responsiveness to ACH were significantly smaller than in group B without a latency period. They were comparable to those of group A. In summary, HDI-induced airway hyperresponsiveness to ACH in the guinea pig is reversible within 8 weeks of HDI avoidance. It is assumed that the augmented airway responsiveness indicates an increased risk of developing isocyanate-induced obstructive lung diseases.

Toluene Diisocyanate Induction of Airway Hyperresponsiveness at the Threshold Limit Value (10 ppb) in Rabbits

Induction of acute lung injury and the development of airway hyperresponsiveness (AHR) by toluene diisocyanate (TDI) exposure was studied in a new rabbit model of occupational lung diseases. TDI in the range of the threshold limit value (TLV) of 10 ppb, as well as at 5 and 30 ppb, administered four times over period of 1 h to three groups of eight rabbits, did not significantly alter airway resistance (RI), dynamic elastance (Edyn), slope of inspiratory pressure generation (ΔPes/tI), arterial pressure (Pa) or aterial blood gas tensions (PaO2, PaCO2). Airway responsiveness (AR) to aerosols of 2% acetylcholine (ACH) was measured before and after each TDI exposure. After TDI inhalation of 10 ppb over 4 h, the amplitude of the ACH-induced airway constrictor response indicated by the changes in Edyn rose significantly to almost twice the control response value (p < 0.005). Similar changes in the amplitude of RI and in the slope of ΔPes/tI were obtained. After inhalation of 5 ppb TDI, no changes in airway reactivity were observed. The responses of respiratory mechanical parameters to ACH rose to three to four times the control responses after exposure to 30 ppb TDI. In a control group of eight animals not undergoing TDI exposure, no significant changes of respiratory responses were obtained after inhalation of 0.2% ACH for 1 min. In summary, TDI atmospheres in the range of TLV increased AR to ACH within 4 h of exposure in this rabbit model. This augmented AR may indicate an increased risk for the development of isocyanate-induced obstructive lung diseases.

Role of sensory neuropeptides in PIV-3-infection-induced airway hyperresponsiveness in guinea pigs

Viral respiratory tract infections are known to induce transient airway hyper-responsiveness. The role of the nonadrenergic noncholinergic neuropeptide system on virus-induced airway hyperresponsiveness was studied in the guinea pig. Ten guinea pigs were inoculated with parainfluenza 3 virus (PIV-3.2 x 10(6) PFU) by nasal route. 16 animals served as untreated controls. Viral infection was proven by histological changes and by demonstration of viral antigen using immunohistochemical techniques. Four days after inoculation, airway responsiveness to inhaled acetylcholine (ACH) aerosol was measured in anesthetized and tracheotomized guinea pigs. The ACH concentration which produced an increase of 100% in pulmonary resistance (PC100 RI) and in dynamic elastance (PC100 Edyn) was calculated from a 5-step ACH dose-response curve (0.125, 0.25, 0.5, 1.0 and 2.0% ACH). Two further groups of 8 PIV-3-infected guinea pigs and 8 noninfected control animals were pretreated with capsaicin in increasing doses (50, 100, 125 and 150 mg/kg) on 4 consecutive days starting 6 days before virus inoculation. Measurements of airway responsiveness to ACH were performed 4 days after virus inoculation. Another 5 uninfected control animals were pretreated only with the solvent for capsaicin and inoculated with virus-free cell supermatant. PIV-3 infection increased airway responsiveness to ACH compared to noninfected controls [PC100 RI 0.81 vs. > 2.0% ACH (median). p < 0.002 PC100 Edyn 0.52 vs. 1.07% ACH (median), p < 0.01]. In capsaicin-pretreated PIV-3-infected animals, airway hyperresponsiveness was completely prevented compared to the virus-infected group without capsaicin pretreatment (PC100 RI > 2.0 vs. 0.81% ACH, p < 0.01; PC100 Edyn 1.42 vs. 0.52% ACH p < 0.01). As neuropeptide depletion with capsaicin completely prevented the increase in airway constrictory response to ACH following virus infection, we conclude that neuropeptides are effectively involved in PIV-3-induced airway hyperresponsiveness in the guinea pig.

Ultrastructural Study of Rabbit Lung Tissue Subsequent to Capsaicin Treatment over Several Days

The neurogenic basis of nonspecific airway hyperresponsiveness (NAH) is poorly understood. Under experimental conditions isocyanates can elicit bronchial hyperresponsiveness in animals. The purpose of our study was to determine whether reactions of neurosecretory granules in nonmedullated C fibers might play a role in NAH. Our experiments were based on the fact that capsaicin treatment causes depletion of neurosecretory granules in vicinity of C fibers. We gave rabbits repeated subcutaneous injections of capsaicin. The animals were then treated with toluene diisocyanate (TDI), inducing airway hyperresponsiveness upon acetylcholine (ACH) inhalation. In capsaicin-treated animals the neurosecretory granules were not evident ultrastructurally and airway hyperresponsiveness did not occur in response to TDI treatment. Controls that were not treated with capsaicin displayed both neurosecretory granules ultrastructurally as well as airway hyperresponsiveness to ACH. We conclude that in the rabbit, NAH is related to the presence of neurosecretory granules adjacent to nonmedullated C fibers.

Subchronic exposure to diisocyanates increases guinea pig tracheal smooth muscle responses to acetylcholine.

Objective: In order to study the threshold concentrations of isocyanates (IC) for induction of lung disorders, constrictive responses of tracheal smooth muscles to acetylcholine (ACH) in guinea pigs with and without diisocyanate [toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI) and diphenylmethane diisocyanate (MDI)] exposure were investigated. Methods: An IC-induced increase in smooth muscle responsiveness was studied by measuring cumulative ACH dose responses (10–10 to 10–4 M ACH). Basal ACH dose-response curves, measured twice in intervals of 1 h using tracheal preparations of 11 guinea pigs previously not exposed to IC, were reproducible. Results: Subchronic in vivo exposures to TDI, HDI, and MDI atmospheres of 10 and 20 parts per billion (ppb) on 5 consecutive days led to significantly (p < 0.05) increased ACH responsiveness of tracheal smooth muscle, whereas concentrations of 2.5 and 5 ppb were not effective. Exposure to HDI atmospheres of 10 ppb for 1, 2, 4, or 8 weeks resulted in a time-dependent increase in ACH responses (p < 0.05) of guinea pig tracheal smooth muscle. Increased tracheal muscle responses to ACH were transient since tracheal preparations from animals exposed to 10 and 20 ppb MDI for 4 weeks and with an exposure-free interval of 8 weeks before preparation did not show enlarged ACH responses, which were present in preparations at the end of the exposure period (p < 0.05). Exposure to low IC concentrations as present in workplaces cause increased ACH responsiveness of guinea pig tracheal smooth muscle. The increased responsiveness of the airways seems to be largely reversible, since normal responses were found after 8 weeks of IC avoidance. Conclusion: Reversibility of IC-induced airway hyperresponsiveness is of great occupational and preventive medical importance. Workers with acquired airway hyperresponsiveness might escape lung damage if the changes are detected in an early stage before alterations in lung function are in a chronic stage.