Oral administration of transgenic barley expressing a Culicoides allergen induces specific antibody response.

BACKGROUND: Insect bite hypersensitivity is an immunoglobulin (Ig)E-mediated dermatitis of horses initiated by bites of midges of the genus Culicoides. Culicoides spp. are not indigenous to Iceland and the prevalence of insect bite hypersensitivity is much higher in horses born in Iceland and exported as compared to Icelandic horses born in a Culicoides rich environment. Immunotherapy is therefore needed. OBJECTIVES: The aim of the study was to express an allergen from Culicoides in barley grain and investigate whether an immune response could be obtained in healthy Icelandic horses by oral treatment with transgenic barley expressing the allergen. STUDY DESIGN: In vivo experiment. METHODS: The allergen was expressed in barley grain with the Orfeus technique. A device was developed to treat horses orally with barley flour. Four Icelandic horses were treated with transgenic barley and 3 with control barley, in total 500 g in 7 feedings. Serum and saliva samples were collected for measuring specific antibodies. RESULTS: The allergen Cul n 2, a hyaluronidase originating from the salivary gland of Culicoides nubeculosus, was expressed in barley. Horses treated with the transgenic barley mounted a Cul n 2 specific IgG1 and IgG4/7 response in serum and saliva. The serum response was significantly different between the transgenic and control barley treated horses for both subclasses and the saliva response for IgG1. The induced serum antibodies bound to the corresponding allergen from Culicoides obsoletus, rCul o 2 and were able to partially block binding of Cul n 2 as well as Cul o 2 specific IgE from insect bite hypersensitivity affected horses. MAIN LIMITATIONS: Small number of horses. CONCLUSION: This study shows that specific antibody response can be induced in horses not exposed to Culicoides, using oral treatment with transgenic barley expressing an allergen. Further studies will determine whether this approach is a useful alternative for prevention and treatment of equine insect bite hypersensitivity.

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells.

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

Comparison of the effects of tamoxifen and toremifene on rat hepatocarcinogenesis.

The hepatoproliferative and cytochrome P450 enzyme inducing effects of two antiestrogens, tamoxifen and toremifene, were compared in female Sprague-Dawley rats using immunohistochemical staining methods. Equimolar doses of the antiestrogens (tamoxifen 45 mg/kg and toremifene 48 mg/kg) were given by oral administration to 6-week-old rats for 12 months including a 3-month recovery period. Controls received the vehicle carboxymethylcellulose. Altogether 90 rats were used in the study. Five rats per dose group were killed after 14 days, 5 weeks, 3, 6 and 12 months of treatment as well as after the 3-month recovery period. Hepatocellular carcinoma was found in four out of five rats after 12 months of tamoxifen treatment. After the 3-month recovery period all tamoxifen-treated rats had large liver tumors (diameter up to 3 cm). No tumors were observed in toremifene-treated rats. Liver cell proliferation was measured by the index of proliferating cell nuclear antigen (PCNA) expression. Immunohistochemical staining with the placental form of glutathione S-transferase (GST-P) was used as a marker for preneoplastic foci. Cytochrome P450 induction was measured using specific antibodies to isoenzymes. Tamoxifen increased the incidence of GST-P-positive foci significantly by 3 months of treatment but toremifene did not as compared with the controls. Liver cell proliferation increased significantly only in the liver tumors of tamoxifen-treated rats after 12 months of treatment and during the recovery period. Both antiestrogens induced the isoenzymes CYP2B1/2 and CYP3A1 within 14 days although tamoxifen was a more powerful inducer. Immunohistochemistry of rat liver sections showed a centrilobular localization of these induced enzyme proteins. The expression of CYP2B1/2 and 3A1 could also be observed in foci after 3 and 6 months of administration and in liver adenomas and in some carcinomas after 12 months of administration with tamoxifen. The results show that tamoxifen, but not toremifene, has the potential to induce and promote the development of rat hepatocarcinogenesis in this experimental model.

The effect of toremifene on bone and uterine histology and on bone resorption in ovariectomised rats.

The effect of the selective oestrogen receptor modulator, toremifene, to inhibit ovariectomy-induced bone loss was studied in rats. The oral doses were 0.3, 3.0 or 30 mg/kg/day for 2 months. 17beta-oestradiol (5 microg/kg/day, subcutaneously) was used as positive control. One group was also treated with a combination of 17beta-oestradiol (5 microg/kg) and toremifene (3.0 mg/kg). Biochemical markers were urinary hydroxyproline and calcium (adjusted with urinary creatinine levels) and the serum level of pyridinoline cross-linked carboxy terminal telopeptide, a bone specific collagen breakdown product. The femoral and sternal trabecular bone thickness served as histological parameters. Ovarectomy increased the levels of hydroxyproline and pyrodinoline and decreased the trabecular bone thickness compared to the sham-operated control group. This was inhibited by both test compounds but 17beta-oestradiol was more efficient. Toremifene did not reverse the ovariectomy-induced reduction of urinary calcium but inhibited the 17beta-oestradiol-related increase. When administered together with oestradiol, toremifene did not reverse the positive effect of 17beta-oestradiol on bone, however toremifene reversed the oestradiol-related uterothrophic effects. These findings indicate that the antagonistic features of toremifene dominate in the rat uterus the agonistic properties do in the bone.

DNA adduct formation by tamoxifen and structurally-related compounds in rat liver.

Binding of diethylstilbestrol and four different triphenylethylene derivatives: tamoxifen, toremifene, clomiphene and triparanol to DNA in rat liver, was studied using the 32P-postlabelling method with HPLC-radioactivity detection. Three different modifications of the 32P-postlabelling technique (a) a bisphosphate method with adduct enrichment by nuclease P1 (NP1)-treatment or (b) by butanol extraction and (c) a monophosphate method, were applied in order to provide an unbiased analysis of adduct formation. When tamoxifen was administered by daily gavage for 4 weeks (80 mumol/kg for 2 weeks and 40 mumol/kg for a further 2 weeks) two major adducts and about six minor adducts were produced in the liver of female Sprague-Dawley rats. Equimolar doses of toremifene produced one apparent adduct. The adduct levels in the tamoxifen and toremifene treated rats were 600 and 2/10(8) nucleotides, respectively. Under conditions used, clomiphene, triparanol and diethylstilbestrol did not produce DNA adducts. The present and previous data suggest that modification (a) is the 32P-postlabelling method of choice for risk assessment in human subjects. Modification (c) with butanol extraction after labelling has the advantage of low background radioactivity and may be preferable if large amounts of DNA are available. The main tamoxifen adducts were suggested to be alpha-(N2-deoxyguanosinyl)tamoxifen and alpha-(N2-deoxyguanosinyl)-N-desmethyltamoxifen.

DNA binding of tamoxifen and its analogues: identification of the tamoxifen-DNA adducts in rat liver.

DNA binding of tamoxifen and some structurally-related drugs (toremifene, clomiphene, triparanol and raloxifene) in rat liver was studied using the 32P-postlabelling method. As only tamoxifen was shown to form high levels of DNA adducts, the identity of these adducts was studied. Recently, we have identified by mass spectroscopy the two main tamoxifen adducts in rat liver DNA as the N-desmethyltamoxifen and tamoxifen adducts of N2-deoxyguanosine in which the linkage is through alpha-carbon in the tamoxifen structure. Minor adducts were identical to different diastereomers of alpha-(N2-deoxyguanosinyl)tamoxifen and of alpha-(N6-deoxyadenosinyl)tamoxifen. Altogether these adducts accounted for at least 95% of adducts formed in vivo, implicating that the alpha-hydroxylation of the ethyl group is the major activation pathway for DNA adducts.

Antioxidant properties of the triphenylethylene antiestrogen drug toremifene.

The aim of the present study was to investigate antioxidativity of the triphenylethylene antiestrogen toremifene. Toremifene and its structural analogues were studied for their ability to inhibit chain reactions of lipid peroxidation and to act as scavengers of free radicals in vitro, and the effects of toremifene were compared to those of the estrogens, tamoxifen and known antioxidants. Moreover, the in vivo antioxidativity of toremifene was tested in a long-term experiment with rats. The ability of toremifene to prevent lipid peroxidation was assayed in two different test systems. In the first assay (initiated with ascorbate/ADP-FeCl3, detection by the formation of TBA-reactive material) toremifene was found to act as an efficient membrane antioxidant with an IC50-value (18 microM) comparable to that of tamoxifen (26 microM) and alpha-tocopherol (43 microM). Toremifene derivatives 4-hydroxytoremifene (IC50 = 8 microM) and Fc 1159 (IC50 = 31 microM), as well as diethylstilbestrol (IC50 = 17 microM) were also active while estradiol showed only weak antioxidativity (IC50 = 300 microM) in this test system. In the other assay (peroxidation initiated with t-butylhydroperoxide, detection by luminol-enhanced chemiluminescence) toremifene prevented lipid peroxidation only at high concentrations (IC50 = 450 microM) but the metabolite 4-hydroxytoremifene (IC50 = 0.18 microM), estradiol (IC50 = 4.6 microM) and diethylstilbestrol (IC50 = 1.7 microM) showed potent antioxidant activity. The potency of 4-hydroxytoremifene even exceded that of alpha-tocopherol (IC50 = 2.0 microM) and butylated hydroxyanisole (IC50 = 1.1 microM). Toremifene was found to have some superoxide anion but no peroxyl radical scavenging activity. Interestingly, diethylstilbestrol turned out to be a potent scavenger of peroxyl radicals. Treatment of female Sprague-Dawley rats with toremifene (12 or 48 mg/kg) was found to decrease serum levels of lipid peroxides. This was seen at various time points (2 days, 5 weeks, 6 and 12 months) during long-term administration of toremifene to rats, and results obtained with two different methods (diene conjugation, TBA-reactive material) gave similar results. The present study thus showed that (i) like steroidal estrogens and tamoxifen toremifene is a potent membrane antioxidant in vitro, (ii) the antioxidant action of toremifene is not due to scavenging of free radicals and, importantly, (iii) toremifene acts antioxidatively also in living organisms in vivo.

A two-year dietary carcinogenicity study of the antiestrogen toremifene in Sprague-Dawley rats.

The carcinogenic potential of the nonsteroidal triphenylethylene antiestrogen toremifene (Fareston) was evaluated in a standard 104-week rat dietary carcinogenicity study. The doses were 0, 0.12, 1.2, 5.0 and 12 mg/kg/day and the number of animals 50/sex/dose group. The body weight gain and food consumption were monitored once weekly (study weeks 1-16) or once every four weeks thereafter (study weeks 17-104). Blood samples were taken at weeks 34, 52 and 104 and the plasma concentrations of toremifene, as well as the two main metabolites (deaminohydroxy)toremifene and N-demethyltoremifene, were measured. All doses of toremifene reduced food intake and body weight gain. Toremifene caused a significant reduction in mortality, which was mainly due to reduced incidences of pituitary tumors. This was evident in all dose groups. Drug-related decrease of mammary tumors in females (at all doses) and testicular tumors in male rats (doses > or = 1.2 mg/kg/day) were also evident. The incidence of the preneoplastic foci of basophilic hepatocytes were significantly decreased in treated female groups. Toremifene induced no preneoplastic or neoplastic lesions. Based on histopathology, no obvious toxicity could be observed. Drug-related changes were observed in the genital organs, thyroid, spleen, mammary gland, adrenal, kidney, stomach and lung. These changes were due to hormonal disturbances or as a result of reduced food consumption or reduced incidences of pituitary, mammary or testicular tumors. This study indicates that toremifene is an efficient antiestrogen in long-term treatment, is well tolerated and has no tumorigenic potential in rats.

Simple methods of quantifying oxidation products and antioxidant potential of low density lipoproteins.

OBJECTIVES: The present study describes new methods for the measurement of oxidation products and antioxidant potential of low density lipoproteins (LDL). DESIGN AND METHODS: LDL is isolated by precipitation with buffered heparin. The assay for LDL oxidation products (LDL-BDC) is based on determination of baseline levels of conjugated dienes (BDC) in lipids extracted from LDL. The assay for antioxidant potential of LDL (LDL-TRAP) is based on the ability of LDL to trap peroxyl radicals. RESULTS: LDL-BDC was found to increase linearly over a range from 100 to 1750 microL, LDL-TRAP from 250 to 1750 microL of serum taken for precipitation. For LDL-BDC, the CV was 4.4% and 4.5% for within- and between-assay precision, respectively. For the LDL-TRAP, the CV was 8.1% and 8.7% for within- and between-assay precisions, respectively. Freezing of the serum (2 weeks at -70 degrees C) did not affect LDL-BDC or LDL-TRAP levels. A negative correlation was found to exist between the LDL-BDC and LDL-TRAP values. LDL-BDC and LDL-TRAP values were at the same level in both sexes. The LDL-BDC was found to increase with age. Short-term intervention with antioxidants increased LDL-TRAP substantially, but did not affect the LDL-BDC level. CONCLUSIONS: These methods are fast and simple to perform, and can, therefore, be applied to clinical purposes.

Tamoxifen and toremifene lower serum cholesterol by inhibition of delta 8-cholesterol conversion to lathosterol in women with breast cancer.

PURPOSE: Long-term effects of tamoxifen and toremifene, a new antiestrogen that closely resembles tamoxifen, were investigated on serum lipids and cholesterol metabolism. PATIENTS AND METHODS: The study group consisted of 24 postmenopausal Finnish women with advanced breast cancer from an international multicenter study of 415 patients. Cholesterol metabolism was evaluated by measuring the cholesterol precursor (delta 8-cholestenol, desmosterol, and lathosterol, reflecting cholesterol synthesis) and plant sterol (markers of cholesterol absorption) and cholestanol levels by gas-liquid chromatography. RESULTS: Tamoxifen and toremifene lowered significantly serum low-density lipoprotein (LDL) cholesterol levels after 12 months of treatment by 16% and 15%, with no change in high-density lipoprotein (HDL) cholesterol or serum triglyceride levels. Serum delta 8-cholestenol was increased 40- and 55-fold during toremifene and tamoxifen treatment, respectively, while the increase of desmosterol less than doubled and was lacking for lathosterol by toremifene. Plant sterols and cholestanol were only inconsistently increased in serum. CONCLUSION: Tamoxifen and toremifene inhibit the conversion of delta 8-cholestenol to lathosterol so that serum total and LDL cholesterol levels are lowered by downregulation of cholesterol synthesis. Thus, inhibition of the delta 8-isomerase may be the major hypolipidemic effect of these agents. Reduced risk of coronary artery disease will probably occur also during long-term toremifene treatment, because the drug reduces cholesterol and its synthesis, similarly to tamoxifen.

Role of Abscisic Acid in Drought-Induced Freezing Tolerance, Cold Acclimation, and Accumulation of LT178 and RAB18 Proteins in Arabidopsis thaliana.

To study the role of abscisic acid (ABA) in development of freezing tolerance of Arabidopsis thaliana, we exposed wild-type plants, the ABA-insensitive mutant abi1, and the ABA-deficient mutant aba-1 to low temperature (LT), exogenous ABA, and drought. Exposure of A. thaliana to drought stress resulted in a similar increase in freezing tolerance as achieved by ABA treatment or the initial stages of acclimation, suggesting overlapping responses to these environmental cues. ABA appears to be involved in both LT- and drought-induced freezing tolerance, since both ABA mutants were impaired in their responses to these stimuli. To correlate enhanced freezing tolerance with the presence of stress-specific proteins, we characterized the accumulation of RAB18 and LTI78 in two ecotypes, Landsberg erecta and Coimbra, and in the ABA mutants during stress response. LT- and drought-induced accumulation of RAB18 coincided with the increase in freezing tolerance and was blocked in the cold-acclimation-deficient ABA mutants. In contrast, LT178 accumulated in all genotypes in response to LT and drought and was always present when the plants were freezing tolerant. This suggests that development of freezing tolerance in A. thaliana requires ABA-controlled processes in addition to ABA-independent factors.

Lipid peroxidation and antioxidant enzyme activities in the rat testis after cigarette smoke inhalation or administration of polychlorinated biphenyls or polychlorinated naphthalenes.

Lipid peroxidation products and antioxidant enzyme activities were studied in the rat testis following exposures to cigarette smoke, polychlorinated biphenyls (PCBs), or polychlorinated naphthalenes (PCNs). Three hours after a single 1-hour period of smoke inhalation, the levels of fluorescent chromolipids and thiobarbituric acid-reactive species (TBARS) were markedly increased in the testis (+49%, P < 0.01, and +43%, P < 0.05, respectively). Twelve hours after daily smoking for 1 hour, for 1, 5, or 10 days, such an increase was not found. Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), glutathione transferase (GSH-Tr), or hexose monophosphate shunt (HMS) were not affected immediately, 3 hours, or 12 hours after a single smoking session. Twelve hours after smoking for 5 days, the activity of catalase was decreased (-16%, P < 0.05). Smoking exposures had no consistent effects on serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), or testosterone concentrations. Single i.p. injections of PCB or PCN mixtures resulted in decreases in testicular SOD activity 1 day after the exposures (-14%, P < 0.05, and -51%, P < 0.01, respectively). Catalase activity also decreased after both exposures (-30 to -42%, P < 0.05, at days 1-7 after PCB exposure, and -37 to -43%, P < 0.05, at days 3-7 after PCN exposure). Ninety days after the PCN exposure, activities of GSH-Px and GSH-Tr were decreased in the testis (-20%, P < 0.05, and -26%, P < 0.05, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of drug metabolizing and antioxidant enzyme activities during tamoxifen-induced hepatocarcinogenesis in the rat.

The triphenylethylene drug tamoxifen is a hepatocarcinogen in rats, has genotoxic potential and may produce carcinoma of the endometrium in humans, while the structurally closely related toremifene has no carcinogenic or genotoxic potential. We have investigated the effects of long-term treatment with tamoxifen and toremifene on the activities of drug metabolizing and antioxidant enzymes in rat liver. Female Sprague-Dawley rats were dosed with equimolar doses of tamoxifen (11.3 and 45 mg/kg) and toremifene (12 and 48 mg/kg) for 12 months and were killed after 2 days, 5 weeks, 3, 6 and 12 months of treatment. After 12 months most rats treated with the high dose of tamoxifen had hyperplastic nodules and hepatocellular carcinomas, while in rats given toremifene or the low dose of tamoxifen, only foci were observed. A striking observation was strong inhibition of the hexose monophosphate shunt (HMS) by tamoxifen and toremifene, which, except in the group given the high dose of tamoxifen, lasted throughout the treatment period. Both antiestrogens induced susceptibility to oxidative stress, as indicated by decreased hepatic contents of reduced glutathione and by increased peroxidation potential of microsomal preparations. The activity of glutathione S-transferase was permanently induced by the high dose of tamoxifen from 5 weeks onwards and was greater in tamoxifen-induced liver tumors than in corresponding macroscopically normal tissue. Similarly, the activity of HMS was elevated by the high dose of tamoxifen at the latest time points, and a further elevation was seen in tamoxifen-induced liver tumors. No such alteration in glutathione S-transferase or HMS activity was seen in animals treated with toremifene or with the low dose of tamoxifen. In conclusion, tamoxifen and toremifene differ markedly with respect to production of liver tumors, and this difference in hepatocarcinogenic potential is reflected in differential effects on glutathione-S-transferase and HMS activities in rat liver.

Alterations in Water Status, Endogenous Abscisic Acid Content, and Expression of rab18 Gene during the Development of Freezing Tolerance in Arabidopsis thaliana.

Treatments as diverse as exposure to low temperature (LT), exogenous abscisic acid (ABA), or drought resulted in a 4 to 5[deg]C increase in freezing tolerance of the annual herbaceous plant Arabidopsis thaliana. To correlate the increase in freezing tolerance with the physiological changes that occur in response to these treatments, we studied the alterations in water status, endogenous ABA levels, and accumulation of rab18 (V. Lang and E.T. Palva [1992] Plant Mol Biol 20: 951-962) mRNA. Exposure to LT and exogenous ABA caused only a minor decline in total water potential ([psi]w), in contrast to a dramatic decrease in [psi]w during drought stress. Similarly, the endogenous ABA levels were only slightly and transiently increased in LT-treated plants in contrast to a massive increase in ABA levels in drought-stressed plants. The expression of the ABA-responsive rab18 gene was low during the LT treatment but could be induced to high levels by exogenous ABA and drought stress. Taken together, these results suggest that the moderate increases in freezing tolerance of A. thaliana might be achieved by different mechanisms. However, ABA-deficient and ABA-insensitive mutants of A. thaliana have impaired freezing tolerance, suggesting that ABA is, at least indirectly, required for the development of full freezing tolerance.

Dietary fat- and phenobarbital-induced alterations in hepatic antioxidant functions of mice.

Inbred strains of mice with differential response to known tumor promoters were compared with respect to their susceptibility to modulation of hepatic antioxidant enzymes by long-term treatment with high fat diet (HF) and phenobarbital (PB). Mice of the C57BL/6J (C57), C3H/HeOuJ (C3H) and DBA/2J (DBA) strains were fed diets containing low (5%) or high (15%) amounts of fat (sunflower oil) for 26 weeks from the age of 6 weeks onwards. Groups of mice on the 5% fat diet received 0.05% PB in their drinking water from 12 to 22 weeks of age. Mice of the C57 strain are known to be refractory to promotion of hepatocarcinogenesis, the C3H strain has a high incidence of spontaneous tumors and is sensitive to promotion by HF and PB, and the DBA strain is especially sensitive to promotion by PB. Within all strains of mice, and in both dietary groups, the degree of oxidative stress in the liver was found to increase with age, as was indicated by the increased amounts of TBA reactive material (lipid peroxidation) and decreased glutathione (GSH) and phospholipid contents of the tissue. HF elevated the amount of TBA reactive material in the liver of C57 and C3H mice, induced GSH-peroxidase and Mn-superoxide dismutase activities in the C3H strain, and depressed the hexose monophosphate shunt activity within all mouse strains. PB drastically decreased the amount of TBA reactive material in the liver in all mouse strains, increased catalase activity in all strains and the activity of GSH-peroxidase in the C3H and DBA strains. The above strain differences in responses of hepatic antioxidant functions to HF and PB parallel the differential responsiveness of these mouse strains to promotion of hepatocarcinogenesis by these agents, and the increased antioxidant capacity was proportional to susceptibility to tumor promotion.

Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus.

Intracerebral serial passage of visna virus KV1514 through three Icelandic sheep was used to select for strains with increased neurovirulence. A strain (KV1772) with increased neuropathogenicity was obtained. We isolated several proviral molecular clones from a plaque-purified biological clone of KV1772 that induced typical visna virus pathology in young sheep. One of the clones (kv72) was infectious, while others contained mutations or were permuted and required gene recombination with other proviral clones to generate infectious virus after transfection. Stable plasmids containing functional, full-length, visna virus KV1772 genomes were constructed from the proviral molecular clones. The in vitro cytopathic effects of virus derived from these clones varied depending upon the tissue origin of the infected cells. A goat cell line became persistently infected with molecularly cloned KV1772 virus; these cells resisted the cell-killing effects and continuously shed high levels of infectious virus. We determined the complete nucleotide sequence of a KV1772 provirus; it contains open reading frames for all structural and accessory genes previously identified in the visna virus genome and is highly homologous to other published visna virus sequences. Progeny of molecularly cloned KV1772 virus rapidly induced both a pronounced neuropathology and an unexpected, strong, neutralizing antibody response in experimentally infected young Icelandic sheep. The availability of stable plasmids of replication-competent and pathogenic proviral molecular clones of visna virus should now enable the study of the genetic determinants of neurovirulence and their interaction with the host immune system in visna virus pathogenesis.

Pro-oxidant effects of normobaric hyperoxia in rat tissues.

Rats were exposed to 100% O2 atmosphere for 12, 36 or 48 h, and their lungs, brain, liver and kidneys were studied for signs of oxidative damage. Oxidative damage at molecular level was estimated by: (1) the appearance of conjugated diene double bonds and (2) the amount of fluorescent chromolipids in lipids extracted from tissues. As important intracellular regulators of oxidative stress, the response of enzymes detoxifying reactive oxygen species was also studied. Macroscopically, the brain and the lungs were most susceptible to oxygen-induced effects. As an indication of oxidative tissue damage, hyperoxia caused accumulation of fluorescent chromolipids in brain and lung tissues, whereas diene conjugation did not reveal any signs of lipid peroxidation. Accumulation of fluorescent chromolipids was most prominent in the brain, where 99 and 138% increases over the control were detected after 36 and 48 h hyperoxia, respectively. Fluorescent chromolipids appeared in urine already before their concentrations were elevated in tissues. The activity of superoxide dismutase in the brain was initially decreased, followed then by a slight induction of activity at the later time-points. Pulmonary and hepatic catalase activities were markedly decreased after prolonged (36 and 48 h) hyperoxia. In conclusion, fluorescent chromolipid formation seems to be a sensitive indicator of hyperoxia-induced oxidative damage in rat tissues. The lipid peroxidation-derived fluorescent chromolipids are eliminated from the body via urinary excretion. Moreover, impaired detoxication of reactive oxygen may be implicated in tissue damage due to hyperoxia.

Enhanced peroxisomal beta-oxidation of fatty acids and glutathione metabolism in rats exposed to phenoxyacetic acids.

Peroxisomal beta-oxidation of fatty acids and the activities of glutathione-metabolizing enzymes in rat liver were measured after administration of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), clofibrate [ethyl], 2-(p-chlorophenoxy)-2-methylpropionate], glyphosate (N-phosphonomethyl glycine, a herbicide not structurally related to phenoxy acids) or saline for 14 days. beta-Oxidation increased by 6-fold in the group given clofibrate, 3-fold in the 2,4-D-treated group, and 2-fold in the MCPA-treated group over the level in the controls (saline-treated). Glyphosate did not increase beta-oxidation. No significant change in reduced glutathione content from that in controls was found in any of the treated groups. Glutathione reductase activity increased by about 40% after administration of either 2,4-D or MCPA, and glutathione peroxidase activity increased by 30% in animals given MCPA. A slight decrease in glutathione S-transferase activity was found in the group treated with clofibrate. The marked increases in peroxisomal beta-oxidation of fatty acids were accompanied by only minor changes in the activities of enzymes involved in glutathione-dependent inactivation of organic hydroperoxides and other oxygen-centred reactive agents.