Influence of ciclosporin on epidermal proliferation after mechanical skin irritation

In mice (NMRI and hr/hr), ciclosporin, subcutaneously administered once daily over 7-10 days, influenced epidermal hyperproliferation induced by abrasion of superficial epidermal layers. While in vehicle-treated control animals the mechanical irritation of skin caused an increase in 3H-thymidine triphosphate incorporation rate into DNA and also in epidermal thickness of tail and flank skin, the hyperproliferative response was diminished in ciclosporin-treated animals. Thus, only a slight increase of the 3H-thymidine triphosphate incorporation rate and of epidermal thickness was measured after pretreatment with ciclosporin, the effect being dose-dependent. The strongest inhibitory activity was found after application of 30 mg/kg BW. These findings indicate that the immunosuppressive peptide ciclosporin inhibits the epidermal proliferation in vivo.     

Inhibition of n-hexadecane-induced epidermal hyperplasia due to systemically administered ciclosporin

Epidermal hyperplasia was induced in hairless mice (hr/hr) by topical n-hexadecane treatment of tail and back skin. Following this skin irritation, a granular layer developed in interfollicular regions of the tail epidermis. An increase of ornithine decarboxylase activity, of thymidine triphosphate incorporation into DNA and of amino acid incorporation into protein was found. Shown histologically and by measurement of the called biochemical parameters, ciclosporin (cyclosporin A, CAS 59865-13-3; pretreatment with 30 mg/kg b.w. per day subcutaneously for 7 days) inhibited the development of epidermal hyperplasia in back and tail epidermis.     

Inhibitory effects of azelastine on substance P-induced itch-associated response in mice

The anti-pruritic mechanisms of azelastine were studied in mice. Scratching induced by intradermal histamine was inhibited by azelastine (30 mg/kg) and chlorpheniramine (30 mg/kg). Substance P-induced scratching was dose dependently suppressed by azelastine (3-30 mg/kg), but not by chlorpheniramine (10 and 30 mg/kg). Azelastine (30 mg/kg) inhibited the substance P-induced production of leukotriene B4, but not prostaglandin E2, in the skin. Azelastine (3-30 mg/kg) suppressed scratching induced by intradermal injection of leukotriene B4. The results suggest that inhibition of the production and action of leukotriene B4, as well as an anti-histamine action, is involved in the anti-pruritic action of azelastine.     

Effects of etretinate in epidermal metabolism and histology after abrasio of superficial layers. Mouse tail assay

The mouse tail assay is suitable for the histological detection of altered differentiation of the epidermis. In the present study, the stratum corneum of the tail epidermis of female mice (NMRI) was removed by means of fine sandpaper. One group of mice was treated with 2 mg of ethyl (all-trans)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl- 2,4,6,8-nonatetraenoate (etretinate)/kg body weight daily. The untreated group served as control. After removal of the stratum corneum epidermal hyperplasia and formation of a stratum granulosum was found in the controls (peak on the third day). The alterations declined from the fourth day onwards. The histidine incorporation into protein was increased from days 4 to 6. A similar reaction sequence was observed in the animals treated with etretinate. In addition to the hyperplasia, there was an enlargement of the cells (hypertrophy). The control group showed a significant elevation of leucine incorporation and a reduction of histidine incorporation into protein on the first day after removal of the stratum corneum. The incorporation rate of thymidine triphosphate into DNA peaked on the second day. The rise of leucine incorporation into protein and the increase of thymidine triphosphate incorporation into DNA on the second day after removal of the stratum corneum could not be detected in the animals treated with etretinate. In these animals histidine metabolism could not be influenced by etretinate. In conclusion, etretinate greatly influences the alteration of basal metabolism caused by the removal of the stratum corneum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Azelastine, a new antiallergic/antiasthmatic agent, inhibits PAF-acether-induced platelet aggregation, paw edema and bronchoconstriction

Azelastine is a phthalazinone derivative with a wide spectrum of pharmacologically relevant activities. Since PAF-acether has been considered to be a potent mediator of asthma, azelastine was assayed for its ability to counteract PAF-acether-induced platelet aggregation, paw edema development and bronchoconstriction. Azelastine exerted a concentration-dependent inhibition of PAF-acether-induced platelet aggregation in human platelet rich plasma with an IC50 of 87 mumol/l and was as effective as ketotifen. PAF-acether-induced paw edema was reduced by intraperitoneal administration of azelastine resulting in an ID50 of 14.4 mg/kg after 2 h. By contrast, ketotifen was not able to inhibit edema development up to a dose of 32 mg/kg i.p. Azelastine and ketotifen, administered intravenously, dose-dependently inhibited PAF-acether-induced bronchoconstriction, starting from the dose of 0.01 mg/kg and resulting in ID50s of 0.03 and 0.02 mg/kg, respectively. These results show that azelastine is endowed with a peculiar anti-PAF-acether action, which may be advantageous in its therapeutic use, in the treatment of asthma.     

Effects of drug metabolism inhibitors on butylated hydroxytoluene-induced pulmonary toxicity in mice

The administration of butylated hydroxytoluene (BHT) to mice results in lung cell damage followed by cellular proliferation which was quantitated by measuring the increase in thymidine incorporation into pulmonary DNA. Administration of SKF 525-A or piperonyl butoxide to mice treated with BHT prevented the increase in thymidine incorporation into pulmonary DNA. This effect was dose dependent, with complete protection from 400 mg/kg BHT achieved with 10 mg/kg SKF 525-A or 400 mg/kg piperonyl butoxide. SKF 525-A and piperonyl butoxide completely prevented the BHT-induced increase in pulmonary DNA synthesis even when given 1–2 hr after BHT and a partial protective effect was evident when they were given 6–12 hr after BHT. Pretreatment of mice with cobaltous chloride diminished the BHT-induced increase in thymidine incorporation into pulmonary DNA. Following the in vivo administration of [¹⁴C]BHT, radioactivity was covalently bound to lung, liver, and kidney macromolecules of both mice, which exhibited BHT-induced lung damage, and rats, which did not. The greatest amount of radioactivity was bound to lung tissue from mice. This binding was prevented by the administration of SKF 525-A and was a linear function of the BHT dose within a range of 50–600 mg/kg. Binding to other tissues from the mouse and all tissues examined in the rat was minimal and unaffected by SKF 525-A. These data suggest that a reactive metabolite of BHT rather than the parent compound produces lung damage in mice.     

Inhibition of cysteinyl-leukotriene production by azelastine and its biological significance

Azelastine is a phthalazinone derivative with a wide spectrum of pharmacological activities. Actively sensitized guinea pigs were used to examine the broncholytic effect of azelastine in vivo. Furthermore, the influence of azelastine on the production of arachidonic acid (AA) metabolites was investigated in vitro and compared to the effects of nordihydroguaiaretic acid (NDGA), indomethacin and ketotifen. In vivo, azelastine protected actively sensitized guinea-pigs against ovalbumin-induced bronchospasm with an ID50 of 0.08 mg/kg orally. Ketotifen was similarly active (ID50 = 0.05 mg/kg). Antigen-induced contraction of isolated tracheal rings of sensitized guinea-pigs was concentration-dependently inhibited by azelastine and NDGA with IC50-values of 94.1 and 34.2 mumol/l, respectively. Ketotifen exerted only weak inhibitory activity (18% at 100 mumol/l). The arachidonic acid-induced contraction of isolated guinea-pig tracheal rings was also inhibited both by azelastine (IC50 = 92.6 mumol/l) and NDGA (IC50 = 20.4 mumol/l). Ketotifen was inactive on this model. Antigen challenge of chopped lung tissue from sensitized guinea-pigs resulted in the release of cysteinyl-leukotrienes (LT) which were identified by reversed phase high pressure liquid chromatography (HPLC) as LTD4 and LTE4. The release of cysteinyl-LT from sensitized guinea-pig lung tissue induced by antigen challenge was concentration-dependently inhibited by azelastine (IC50 = 35.2 mumol/l) and NDGA (IC50 = 8.4 mumol/l) but not by ketotifen and indomethacin. By contrast, indomethacin caused a pronounced augmentation of cysteinyl-LT release. The concentration of indomethacin, which augmented cysteinyl-LT release by 50% was 0.19 mumol/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of azelastine hydrochloride, a novel anti-allergic drug, on the central nervous system

The effects of 4-(p-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepin-4-yl)-1-(2H)-phthalazinone hydrochloride (azelastine hydrochloride), a new anti-allergic drug, on the central nervous system were studied in mice, rats and rabbits in comparison to clemastine, chlorpheniramine and diphenhydramine. In mice, azelastine showed a definite central action with the same dose range (10--40 mg/kg p.o.) as the reference drugs. However, the central action profile of azelastine was considerably different from those of the reference antihistaminics. Clemastine and diphenhydramine produced potent anti-tremorine and anti-pilocarpine activities, while azelastine was less active than the two drugs. Chlorpheniramine and diphenhydramine produced a marked anti-reserpine activity and a marked potentiation of the caffeine-induced hypermotility, while clemastine had little effect in this animal model; on the contrary, 40 mg/kg of azelastine suppressed the caffeine-induced hypermotility. In rats, a high dose of 100 mg/kg azelastine produced a slight suppression of the paradoxical sleep. But the drug affected neither the rectal temperature nor the conditioned avoidance response at oral doses up to 100 mg/kg. In unanesthetized and restrained rabbits, azelastine in a dose of 0.5 to 8 mg/kg i.v. showed no significant change in the spontaneous EEG activity and in the susceptibility of the ascending reticular activating system, whereas an i.v. dose of 2 mg/kg clemastine produced a marked high voltage and slow wave pattern in EEG activity and suppressed the reticular activating system.     

Low iron state is associated with reduced tumor promotion in a two-stage mouse skin carcinogenesis model.

The purpose of this study was to investigate the effect of low iron state in a two-stage mouse skin carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and benzoyl peroxide (BPO)-promoted cutaneous tumorigenesis. All mice were treated with DMBA. Low iron state was achieved by injection with phenylhydrazine hydrochloride and feeding low iron diet. A low iron state resulted in a decrease in tumor incidence (papillomas and carcinomas) and number of tumors/mouse. Also, the conversion of papillomas to carcinomas was lower in mice on a low iron state. BPO treatment enhanced epidermal lipid peroxidation (LPO) and was accompanied by a depletion in the level of epidermal reduced glutathione (GSH) and decrease in the activities of antioxidant enzymes. BPO treatment also increased ornithine decarboxylase (ODC) activity and [3H]thymidine incorporation into cutaneous DNA. Mice in a low iron state were less susceptible to the effects of BPO treatment, as was apparent from a partial recovery of GSH levels and the activities of antioxidant enzymes, as well as a lower induction in ODC activity, [3H]thymidine incorporation into cutaneous DNA and lesser epidermal LPO. As expected, cutaneous iron levels were lower in mice on a low iron state. Thus, our data show that the tumor-promoting potential of BPO is reduced by low iron state in a two-stage mouse skin carcinogenesis model.     

Inhibition by germanium oxide of the mutagenicity of cadmium chloride in various genotoxicity assays.

The effects of germanium oxide on the genotoxicity of cadmium chloride were investigated. The incorporation of [3H]thymidine into testicular DNA was inhibited in mice injected ip with 1.35, 1.80 or 2.70 mg cadmium chloride/kg body weight. Germanium oxide (0.05 or 0.1 mg/kg body weight, sc) alone did not affect [3H]thymidine incorporation into testicular DNA but 0.05 mg germanium oxide/kg antagonized the inhibitory effect of 1.35 mg cadmium chloride/kg. However, combinations of the other doses of the two compounds did not show statistically significant antagonistic effects. Cadmium chloride significantly increased the frequencies of micronucleus formation in polychromatic erythrocytes, and of chromosome aberrations in the bone marrow of mice treated with 0.7, 1.4 or 2.7 mg/kg body weight, in a dose-related manner. These effects were inhibited by germanium oxide at doses of 0.1 or 0.5 mg/kg body weight, although germanium oxide alone did not affect micronucleus formation or the chromosome aberration rate. Cadmium chloride produced a dose-related increase in the frequency of sister chromatid exchanges in cultured human lymphocytes at concentrations of 5, 10 or 50 mumol. This effect was also inhibited by germanium oxide (0.05 or 0.1 mumol), although germanium oxide alone had no effect. There was a dose-related increase in the frequency of sperms with abnormal head morphologies from mice treated with 0.6, 1.1 or 2.2 mg cadmium chloride/kg body weight and this too was antagonized by the injection of germanium oxide (0.1 or 0.5 mg/kg body weight). Germanium oxide alone did not affect the frequency of sperm-head abnormalities.     

Biochemical and morphologic investigation of the influence of chlorphentermine and subsequent withdrawal on newborn rat lung

Daily oral administration of the anorexigenic drug chlorphentermine (60 mg/kg for 7 days) to 1-day-old rat pups produced an accumulation of hypertrophic macrophages in pulmonary alveoli. Only a few alveoli were found to contain these cells in animals administered 40 mg/kg and there were no significant histopathological alterations in the lungs of animals given 20 mg/kg or physiological saline. A 7-day withdrawal from drug treatment in rat pups previously given 60 mg/kg chlorphentermine for 1 week resulted in a marked reduction in the number of hypertrophic macrophages, and complete disappearance of these cells from the alveoli occurred after a 2-week observation period. Despite the apparent absence of ultrastructural changes, administration of 20 mg/kg chlorphentermine for 7 days significantly enhanced the incorporation of thymidine into lung DNA. This increase was greater with 40 mg/kg and maximal (threefold) with 60 mg/kg. After withdrawal for 2 weeks in neonates previously given chlorphentermine for 1 week at any of these dosage levels, the lungs still incorporated a significantly higher amount of thymidine into DNA as compared to controls. After a 3-week withdrawal period, incorporation of thymidine into pulmonary DNA in all treated groups had returned to approximate control values. These results demonstrate that the chlorphentermine-induced cytological and metabolic alterations are dose dependent in newborn rats and that estimation of incorporation of thymidine into DNA appears to be the more sensitive indicator of chlorphentermine-induced effects on lung.     

Inhibition by azelastine of the effects of platelet-activating factor in lungs from actively sensitised guinea-pigs.

The effect of azelastine on platelet-activating factor (PAF)-induced bronchoconstriction and mediator release in isolated lungs from actively sensitised guinea-pigs was investigated. Guinea-pigs were actively sensitised with two s.c. injections of 10 micrograms ovalbumin in 1 mg Al (OH)3 at a 2-week interval. One week after the second injection, the lungs were removed and challenged intra-arterially with PAF or arachidonic acid. In some experiments lungs from non-immunised guinea-pigs were injected with PAF or histamine. Bronchoconstriction, the release of thromboxane (TX)B2 or leukotriene (LT)-like material and the histamine content of the effluent were evaluated. Azelastine was given s.c. at 10 or 100 micrograms/kg, 4 h before lung removal. Azelastine (100 micrograms/kg) did not inhibit PAF-induced bronchoconstriction and mediator release from lungs from non-immunised guinea-pigs. In contrast, the hyperresponsiveness to 1 ng PAF observed in lungs from actively sensitised animals was dose dependently inhibited by azelastine. Azelastine did not reduce the histamine-induced bronchoconstriction and consequent TXB2 release from lungs from immunised guinea-pigs, indicating that the protective effect exerted against hyperresponsiveness to PAF was not due to histamine antagonism. Azelastine also reduced arachidonic acid-induced bronchoconstriction and LT-like material release from sensitised lungs, regardless of the presence of indomethacin. These results suggest that inhibition of lung hyperresponsiveness to PAF by azelastine may result from an interference with leukotriene synthesis.     

Response of murine epidermal keratinocyte cultures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): comparison of haired and hairless genotypes

Based on the observation that congenic haired and hairless mice differ in the hyperkeratinizing/hyperproliferative epidermal response following topical exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo, it has been proposed that this response in mice segregates with the Ah locus (which controls for the Ah receptors) and the hr locus (for hairlessness) (Knutson and Poland, 1982). In the present studies this hypothesis was tested by comparing the response of epidermal keratinocytes derived from genotypically segregated newborn haired and hairless HRS/J mice to TCDD exposure in in vitro cultures. the parameters monitored were stimulation of cell proliferation (as measured by protein content and rate of tritiated thymidine incorporation in cultures), epidermal transglutaminase activity, cornified cell envelope formation, and keratin staining with Rhodanile blue. Results suggested that the sensitivity of HRS/J haired and hairless mouse epidermal keratinocyte cultures to TCDD exposure in vitro was very similar. In both cell cultures all parameters monitored were stimulated by TCDD exposure in a similar dose-dependent manner. This suggests that physiologic factors beyond the epidermal cells may be involved in expression of the different responses seen in the skin of mice to TCDD in vivo.     

Effect of azelastine on platelet-activating factor and antigen-induced pleurisy in rats.

The interference of azelastine with pleurisy induced by antigen was investigated in actively sensitized rats. The antigenic challenge (ovalbumin, 12 micrograms/cavity) caused early plasma leakage, which peaked within 4 h, accompanied by intense neutrophil infiltration. Pleural exudate decayed 24 h after antigen provocation, when a long-lasting increase in the number of resident eosinophils was observed. Oral pretreatment with azelastine (1-10 mg/kg) dose dependently inhibited the vasopermeation (ED50 = 4.2 mg/kg) and reduced the pleural exudate (ED50 = 6.8 mg/kg) induced by the antigen. In contrast, azelastine (10 mg/kg) failed to modify the neutrophil influx observed at 4 h and the eosinophil accumulation detected at 24 h. Azelastine was also effective against rat pleurisy induced by either platelet-activating factor (PAF-acether), histamine or serotonin. It reduced exudation and the increase in the number of mononuclear cells, neutrophils and eosinophils observed 6 h after PAF-acether. Nevertheless, antagonism of PAF-acether may not be relevant to the inhibition observed in the present model of allergic pleurisy, as the inhibition was refractory to three distinct PAF-acether receptor antagonists. In contrast, like azelastine, the histamine H1 receptor antagonist meclizine and the dual histamine and serotonin receptor antagonist cyproheptadine blocked antigen-induced exudation and failed to interfere with cell influx. We conclude that the anti-exudatory activity of oral azelastine on antigen-induced pleurisy is consistent with it exerting direct effects against vasoactive amines, but is not related to an effect against leucocyte infiltration nor to its ability to inhibit PAF-acether.     

Low iron diet retards 12-<Emphasis Type="Italic">O</Emphasis>-tetradecanoyl phorbol-13-acetate-mediated tumor promotion in murine skin

Recently, we have shown that low iron state reduces benzoyl peroxide-mediated tumor promotion in murine skin. To further test the dependence of iron on skin tumor development, we assessed the effect of a low iron diet on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-mediated tumor promotion in murine skin. Female Swiss albino mice were fed on a low iron diet and initiated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA, 40 µg as150 µl per mouse) and promoted with TPA (2.5 µg as 200 µl per mouse, twice-weekly application for 20 weeks). The appearance of the first papilloma and the number of tumors (papillomas and carcinomas) per mouse were recorded weekly. We observed a decrease in tumor incidence (papillomas and carcinomas) and number of tumors per mouse in TPA-promoted mice fed on low iron diet (0.23 mg Fe/kg diet) compared to normal mice fed on balanced mouse chow (1.70 mg Fe/kg diet). The total iron consumption per day by mice being fed on balanced mouse chow was 340–400 µg Fe/kg body weight, and the total iron consumption per day by mice being fed on low iron diet was 45–55 µg Fe/kg body weight. The number of papillomas per mouse was 45% lower and the conversion of papillomas to carcinomas was about 20% lower in mice fed a low iron diet. The tumor size was also smaller in mice being fed on low iron diet. TPA treatment resulted in decreases in the activities of antioxidant enzymes and depletion in the level of epidermal reduced glutathione (GSH). Feeding mice on low iron diet along with TPA treatment resulted in ~50–75% recovery of the depleted levels of GSH and antioxidant enzymes. In addition, TPA-mediated induction of biological markers of tumor promotion, viz. ornithine decarboxylase activity and [³H]thymidine incorporation into cutaneous DNA, was about 60% lower in TPA-treated mice fed on low iron diet than in normal mice treated with TPA. Cutaneous iron levels were also lower in mice fed on low iron diet than in mice fed on normal diet. Histopathological sections of the skin portion adjoining tumors showed a lower degree of epidermal hyperplasia and lesser infiltration of inflammatory cells in the dermis, and absence of hyperkeratosis in mice fed on low iron diet. Thus, in this study we observe that the tumor promoting potential of TPA is reduced in mice fed on low iron diet, which is also accompanied by lesser inflammatory changes in the skin of tumor-bearing mice fed on low iron diet.     

Effect of TCDD on the density of Langerhans cells in murine skin

Tetrachlorodibenzo-p-dioxin (TCDD) is a prototype for a group of toxic polyhalogenated aromatic hydrocarbons. We have studied the effect of TCDD on skin, specifically the difference in cutaneous response of congenic haired (hr/+) and hairless (hr/hr) mice. Topical application of 0.6 microgram of TCDD induces epidermal hyperplasia/hyperkeratinization in the skin of hr/hr mice, but does not affect the epidermis of congenic hr/+ littermates. Suppression of various parameters of the immune response has been found to be another effect of TCDD exposure in experimental animals. In the present study, we investigated the effect of topical treatment with TCDD on the density of epidermal immune cells, the Langerhans cells (LC), in the skin of hr/hr and hr/+ mice. Results showed that TCDD-induced epidermal hyperplasia/hyperkeratinization in skin of hr/hr mice is accompanied by an increase in the density of LC. In the skin of hr/+ mice, in which TCDD exposure does not induce hyperplastic changes, LC densities are not affected. The increase in LC densities in TCDD-treated hr/hr mouse skin did not result in increased sensitivity of the skin to contact hypersensitization with dinitrofluorobenzene, as measured by changes in ear thickness. When hr/hr murine skin was grafted into skin of hr/+ mice and the entire dorsal skin (including the graft) treated with TCDD, LC were increased in the grafted skin, but not in the surrounding hr/+ skin. Conversly, when hr/+ murine skin was grafted into hr/hr mice and both treated with TCDD, there was no increase in the density of LC in the grafted hr/+ skin. Concomitant treatment of hairless mice with TCDD and with indomethacin did not affect the increase in the density of LC induced by TCDD treatment alone. These findings suggest that TCDD-induced epidermal changes in hr/hr murine skin involve production of factors which mediate the increase in epidermal LC.     

Trichloroacetate stimulation of liver DNA synthesis in male and female mice

Male and female B6C3F1 mice were given trichloroacetate (TCA) by gavage for 11 days. Livers from untreated male and female mice were unremarkable by histopathologic examination. In livers from mice receiving 1000 mg/kg body weight, the centrolobular hepatic cords showed slight changes, which included increased eosinophilic staining and rare apoptosis. Areas in the intermediate zone were noted where the architecture of the liver hepatic cords was subtly changed. The changes in cord architecture seemed to define nodular areas where cellular proliferation in animals treated with TCA had occurred. No histopathologic differences were noted between the livers of treated or control, male and female animals. Mitosis and DNA synthesis were examined using incorporation of ³H]thymidine into liver cells. ³H]Thymidine incorporation into extracted liver DNA of animals receiving TCA was significantly increased over controls in all treatment groups. Autoradiographic examination of liver sections showed that the incorporation of label in control animals was predominantly in peri-sinusoidal cells, whereas the majority of radiolabel incorporation in TCA-treated animals was found in intermediate zone cells that appeared to be mature hepatocytes. No outstanding differences in the distribution of radiolabel in the liver sections from male or female mice were noted. When incorporation of ³H]thymidine was quantified by enumeration of labeled liver cells following autoradiography, incorporation of the radiolabel into hepatocytes increased with the dose of TCA given but there was no increase in radiolabel in peri-sinusoidal cells. Increased mitotic figures in intermediate zone cells resembling mature hepatocytes were noted in all mice treated with TCA. These results suggest that increased DNA synthesis and mitosis may contribute tumorigenesis by TCA.     

O,S,S,-trimethyl phosphorodithioate-induced lung damage in rats and mice

O,S,S,-Trimethyl phosphorodithioate (OSS) is a contaminant of various organophosphorus insecticides which induces delayed damage to rat lung bronchiolar and alveolar epithelial cells. Whether lung damage occurs in mice has not been tested. Changes in DNA synthesis, an index of cell division after the induction of damage, were monitored by measuring thymidine incorporation into pulmonary DNA. Mice, treated with 45 mg/kg OSS, exhibited a significant increase in pulmonary thymidine incorporation on Day 5. Maximal increases occurred on Days 7-10 and were followed by a gradual decline to control levels by Day 15. The labeling index of mouse lung cells, determined following autoradiography, exhibited a similar time course. Differential cell counts indicated that maximal division of type II cells occurred before that of interstitial cells, although interstitial cells were the predominant type labeled at all times. Pulmonary DNA synthesis was significantly increased in rats 2 days after treatment with 90 mg/kg OSS. Maximal thymidine incorporation was measured on Day 3, followed by a decline to control levels on Day 5. Thymidine incorporation into total lung DNA was dose related in both species. Maximal increases appeared after 45 and 90 mg/kg OSS in mice and rats, respectively. The histopathological changes in mouse lung tissue were similar, but somewhat less severe than those seen in rats. Rats exhibited a severe interstitial pneumonitis with type I alveolar cell destruction followed by type II cell proliferation. Mice exhibited a mild to moderate alveolitis with only slight damage to type I cells. Necrosis of bronchiolar Clara cells was evident in both species but was more extensive in rats. SKF 525a and piperonyl butoxide prevented OSS-induced increases in pulmonary DNA synthesis in rat lung suggesting that metabolic activation was necessary to elicit damage. Piperonyl butoxide treatments had no effect, however, on thymidine incorporation after OSS in mouse lung tissue, and the highest dose of SKF 525a had only a moderate inhibitory effect on this parameter while increasing animal mortality. These data indicate that systemic treatment with OSS results in damage to mouse, as well as rat, lung tissue at both the alveolar and bronchiolar levels.     

The effect of the nucleoside transport inhibitor dipyridamole on the incorporation of [3H]thymidine in the rat

Dipyridamole is a non-specific inhibitor of nucleoside transport into mammalian cells. It is currently undergoing clinical evaluation in combination with various antimetabolites in an attempt to enhance the activity of these anticancer drugs by blocking the salvage of extracellular nucleosides, an important determinant of their cytotoxicity. In the present study, the effect of i.v. infusions of dipyridamole on [3H]thymidine incorporation into DNA has been examined in the anaesthetized rat. The tissues studied were bone marrow, gastrointestinal tract epithelium and the ascitic form of the Walker carcinosarcoma. Dipyridamole at 10 mg/kg, given over 3 hr, led to plasma levels of less than 5 microM and did not reduce [3H]thymidine incorporation into any of the tissues studied. At 40 mg/kg dipyridamole (plasma levels 10-15 microM) [3H]thymidine incorporation into the DNA of bone marrow and gastrointestinal tract epithelium was reduced to 20-30% of control values. Increasing the dose to 100 mg/kg did not lead to a further suppression of incorporation. Measurement of [3H]thymidine plasma pharmacokinetics and the intracellular distribution of tritium suggested that the inhibition of [3H]thymidine incorporation was due to reduced cellular uptake. In contrast to the effects on normal tissues, even at a lethal dose (200 mg/kg) dipyridamole did not significantly inhibit [3H]thymidine incorporation into Walker tumour cells. The levels of dipyridamole found in the ascitic fluid, at 100 mg/kg approximately half those in plasma, argue against a pharmacokinetic basis for this difference. Dipyridamole was found to bind extensively (97%) to rat plasma proteins, which may explain the discrepancy between the concentrations of dipyridamole required to inhibit nucleoside incorporation in vitro, in serum-free media, and those needed in vivo. From a comparison of the plasma levels of dipyridamole which cause an inhibition of [3H]thymidine incorporation in the rat with those which can be achieved safely in patients, it is concluded that dipyridamole is unlikely to markedly reduce nucleoside salvage in man.     

Lung growth in mice after a single dose of butylated hydroxytoluene.

In male Swiss-Webster mice, a single injection of butylated hydroxytoluene (BHT) produced a dose-dependent increase in lung weight. Histopathological changes were well developed 3 days after 500 mg/kg of BHT. After 5 days, there seemed to be a proliferation of many alveolar cells, formation of giant cells, and macrophage proliferation. Lower doses of BHT produced similar, although less extensive changes. The histopathologic alterations were accompanied by biochemical changes: 2 days after BHT, there was a significant increase in lung weight and total amounts of DNA, RNA, and protein. The changes were dose-dependent and the smallest effective dose was 250 mg/kg of BHT. Five days after BHT, the highest doses of BHT (500 and 1000 mg/kg) produced a 1.5- to 2-fold increase in lung weight, total DNA, and protein, and a 3- to 4-fold increase in total pulmonary RNA. The incorporation of thymidine into DNA and of leucine into protein increased from 2 days on after BHT. On the other hand, the incorporation of orotic acid into total pulmonary RNA was lower in the treated animals than in the controls. Administration of a single dose of BHT might offer a convenient tool to study the biochemical changes preceding and/or accompanying stimulated cell growth in lung.