Acute, multiple-dose, and genetic toxicology of AR177, an anti-HIV oligonucleotide.

AR177 (Zintevir) is a 17-mer oligonucleotide that has been shown to have anti-HIV activity and to be a potent HIV-1 integrase inhibitor in vitro, and is among the first oligonucleotides to enter human clinical trials. Acute and multiple-dose intravenous toxicity studies were performed in mice, and genetic toxicity studies were performed in vitro and in vivo in order to determine the toxicity profile of AR177. The acute toxicity study in mice showed that AR177 had an LD50 of > or = 1.5 g/kg body weight. The multipledose toxicity study in mice showed that AR177 caused male-specific mortality, and changes in serum chemistry, hematology, and histology at doses of 250 and 600 mg/kg. Clinical chemistry findings included changes in liver function, and decreased erythrocyte values at 250 and 600 mg/kg. Histopathologic findings included vacuolization of reticuloendothelial cells in phagocytic cells in lymphoid tissue, liver, lungs, heart and uterus, and extramedullary hematopoeisis in the spleen. Renal toxicity was exhibited as nephropathy and tubular necrosis in the two high-dose groups of males. A no-effect dose was not established. AR177 did not exhibit genetic toxicity in any of three mutagenic assays. In combination with previously reported toxicity studies of AR177 in monkeys, this study showed that the toxicity of AR177 is species specific.

Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. V: Results with 46 chemicals.

Forty-six coded chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary (CHO) cells using a standard protocol with and without exogenous metabolic activation. Sixteen chemicals were negative and 15 were positive in both assays; 15 were positive for SCEs only (one chemical that was positive for SCEs was equivocal for ABs), and no chemicals induced ABs only. The effect of cell harvest time on the ability to detect the induction of ABs was examined for 18 chemicals. Seven chemicals produced a positive response using both standard and extended harvest times, five were positive only using an extended harvest time, and six were negative using both harvest times. The relationship between cell cycle delay and SCE induction was also examined, and the two appear to be unrelated.

Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro: II. Results with 20 chemicals.

Twenty chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary cells (CHO). These chemicals were tested with and without an added metabolic activation system (rat liver S9 fraction). Four chemicals were negative in both assays, 1 induced ABs only, and 15 were positive for SCEs; 6 of these 15 also induced ABs. The effect of cell harvest time on the ability to detect the induction of chromosomal aberrations was examined for six chemicals. Five of these had caused at least one of the following: cell cycle delay, aberrations observed in first division metaphase cells in the SCE assay, or a weak response in the standard AB assay (10-12-hr growth period). Three chemicals, chlorinated trisodium phosphate, 1,2-dibromo-3-chloropropane, and tetrakis(hydroxymethyl)phosphonium chloride, were positive using both the standard and extended harvest times. N-Nitrosodimethylamine and diphenhydramine HCl were only positive using an extended harvest time, and malonaldehyde was negative using both standard and extended harvest times.

Chromosome aberration and sister chromatid exchange test results with 42 chemicals.

Forty-two chemicals were tested for their ability to induce cytogenetic change in Chinese hamster ovary cells using assays for chromosome aberrations (ABS) and sister chromatid exchanges (SCE). These chemicals were included in the National Toxicology Program's evaluation of the ability of four in vitro short-term genetic toxicity assays to distinguish between rodent carcinogens and noncarcinogens. The conclusions of this comparison are presented in Zeiger et al. [Zeiger E, Haseman JK, Shelby MD, Margolin BH, Tennant RW (1990): [Environ Molec Mutagen 16(Suppl 18): 1-14]. The in vitro cytogenetic testing was conducted at four laboratories, each using a standard protocol to evaluate coded chemicals with and without exogenous metabolic activation. Most chemicals were tested in a single laboratory; however, two chemicals, tribromomethane and p-chloroaniline, were tested at two laboratories as part of an interlaboratory comparison. Four chemicals (C.I. basic red 9 HCl, 2-mercaptobenzothiazole, oxytetracycline HCl, and rotenone) were tested for SCE in one laboratory and in a different laboratory for ABS. Tetrakis(hydroxymethyl)phosphonium sulfate was tested at one laboratory and the chloride form was tested at a different laboratory. Twenty-five of the 42 chemicals tested induced SCE. Sixteen of these also induced ABS; all chemicals that induced ABS also induced SCE. There was approximately 79% reproducibility of results in repeat tests, thus, we conclude that this protocol is effective and reproducible in detecting ABS and SCE.

The effect of a tumor promotor, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on sister-chromatid exchange formation in cultured Chinese hamster cells.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) does not increase the sister-chromatid exchange (SCE) frequency in either Chinese hamster ovary (CHO) or lung (V 79) cells which are cultured in the presence of 5-bromodeoxyuridine. Moreover, TPA does not alter the induction of SCEs in CHO cells by mitomycin C during the first 3 cycles following addition of the alkylating agent. These SCE induction data do not by themselves support the hypothesis that tumor promotion by TPA depends on the enhancement of mitotic recombination.

Nurses' and pharmacists' exposure to antineoplastic drugs: findings from industrial hygiene scans and urine mutagenicity tests.

Data from 83 nurses and pharmacists handling antineoplastic drugs and 35 nurse/pharmacist controls who participated in a national study of antineoplastic drug-handling risks were examined to investigate antineoplastic drug exposure. Measures of external exposure included self-completion drug logs and industrial hygiene scans conducted in clinical settings. Internal exposure was measured by urine mutagenicity tests on end-of-week 24-hour urine specimens. To control for potential confounders, the staff was asked to complete food and hobby diaries and to avoid identified mutagenic substances for 1 week before collection of 24-hour urine samples. On the scans of the drug handlers, 13% showed one or more spots of drug contamination on gloved and ungloved hands, gowns, or shoes. Of the 24-hour urine samples, 15% were mutagenic for Salmonella typhimurium: Rates did not differ significantly for drug handlers and controls. Among nurses who both prepared and administered antineoplastics, those with positive mutagenicity tests handled more doses of the drugs, used less skin protection, and had more skin contact with the drugs than those with negative tests. Nurses who only administered the drugs and had positive mutagenicity tests handled fewer doses of drugs than those with negative tests, but they also reported less use of protection and more skin contact. For both groups of nurses, skin contact with antineoplastics was associated with positive mutagenicity test results (p < 0.01).

A comparison of the effectiveness of pre-hospital bag-valve-mask ventilation performed by Irish emergency medical technicians and anaesthetists working in a tertiary referral teaching hospital.

The objective of this study was to assess the effectiveness of bag-valve-mask ventilation performed by emergency medical technicians with prehospital clinical responsibilities and to compare this with anaesthetists working clinically in tertiary referral teaching hospitals. Participants were asked to perform bag-valve-mask ventilation for three minutes on a Resusi Anne manniquin using a facemask and a two litre self inflating bag. The tidal volumes generated were quantified using a Laerdal skillmeter computer as grades 0-5 corresponding to 0, 335, 434, 561, 673 and > 800 mls. The effectiveness of bag-valve-mask (i.e. the proportion of ventilation attempts which achieved a tidal volume of > 434 mls) was greater for emergency medical technicians [88.2 (17.1)%] than for anaesthetists [40.4 (36.5)%] (P<0.001). Six of the 27 anaesthetists, but none of the 29 emergency medical technicians, were unable to produce even one effective tidal volume i.e. > 434 mls. It is likely that emergency medical technicians are able to perform adequate bag-valve-mask ventilation.

DNA synthesis in competent Bacillus subtilis cells.

Competent cells of Bacillus subtilis incorporate degradation products from transfecting DNA into their chromosomal DNA. The sensitivity of this incorporation to inhibitors of bacterial DNA synthesis [phage infection or 6-(p-hydroxyphenylazo)-uracil] suggests that semiconservative DNA synthesis can occur in competent cells.

Search for DNA interchange corresponding to sister chromatid exchanges in Chinese hamster ovary cells.

Chinese hamster ovary cells (CHO) grown for one cycle in bromodeoxyuridine (BrdU) contain a small amount (0.5%) of unusually dense double stranded DNA. This dense DNA has been previously interpreted as being bifilarly substituted with BrdU and hence evidence that sister chromatid exchange (SCE) formation proceeds via the Holliday model of recombination. However, the amount of this dense DNA is 100 times greater than that expected based on the SCE frequency in similarly cultured CHO cells, and it is not increased by treating the cells with mitomycin C. Moreover, contrary to expectations for bifilary substituted DNA, the amount of this dense DNA is not reduced by growing BrdU-labeled cells for a second cycle in TdR. Finally, DNA isolated from CHO cells contains a minor band (0.5%) with a density 0.025 gm/cc greater than that of the main band, whether or not BrdU has been incorporated. These results call into question the identification of this unusually dense DNA as bifilarly substituted and hence its previously postulated relationship to SCE formation.

Characterization of sister chromatid exchange induction by 8-methoxypsoralen plus near UV light.

The combination of 8-methoxypsoralen and near UV light is highly effective in inducing sister chromatid exchanges (SCEs) in Chinese hamster ovary cells. Appreciable increases in SCEs can be effected by treatments compatible with cell survival, and effects of a single dose of alkylation persist over multiple generations. Both the frequency and location of SCEs induced at different times within the DNA synthesis period varies in a manner indicating that exchange induction is restricted to regions which replicated during or after DNA damage.

Induction of sister chromatid exchanges and gene mutations in Chinese hamster ovary cells by psoralens.

Three linear psoralen compounds, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), 3-carbethoxypsoralen (3-CP), and one angular psoralen, 5-methylangelicin (5-ANG), were tested for their ability to induce both sister chromatid exchanges (SCE) and gene mutations (hypoxanthine-guanine phosphoribosyltransferase locus) in vitro in Chinese hamster ovary cells (CHO line). All the compounds induced both SCE and mutations in the presence of UV irradiation (UVA; peak at 330-380 nm), but no increases were observed in its absence. The frequency of both responses increased with either 1) increasing concentration of compound with a fixed amount of UVA or 2) increasing amount of UVA with a fixed concentration of psoralen. Significant increases in SCE were seen for 8-MOP, 5-MOP, and 5-ANG at concentrations near 1 X 10(-6) M, whereas concentrations near 20 X 10(-6) M of 3-CP were needed before increases in SCE were observed. The induction of gene mutations followed a similar pattern; concentrations of 50-100 X 10(-6) M of 3-CP were needed to induce large increases in mutations, but much lower concentrations of 8-MOP, 5-MOP, and 5-ANG (5-10 X 10(-6) M) were sufficient to induce large increases in mutations. The ratio of induced mutations to induced SCE was similar for 8-MOP, 5-MOP, and 3-CP; that of 5-ANG was much higher, which indicated that the linear furocoumarins produce a different spectrum of DNA damage from that produced by the angular psoralen.

Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction.

Unilateral ureteral obstruction (UUO) is a model of renal injury characterized by progressive tubulointerstitial fibrosis and renal damage, while relatively sparing the glomerulus and not producing hypertension or abnormalities in lipid metabolism. Tubulointerstitial fibrosis is a major component of several kidney diseases associated with the progression to end-stage renal failure. Here we report that when a critical renal developmental morphogen, osteogenic protein-1 (OP-1; 100 or 300 microg/kg body wt), is administered at the time of UUO and every other day thereafter, interstitial inflammation and fibrogenesis are prevented, leading to preservation of renal function during the first 5 days after obstruction. Compared with angiotensin-converting enzyme inhibition with enalapril treatment, OP-1 was more effective in preventing tubulointerstitial fibrosis and in preserving renal function. The mechanism of OP-1- induced renal protection was associated with prevention of tubular atrophy, an effect not shared with enalapril, and was related to preservation of tubular epithelial integrity. OP-1 blocked the stimulation of epithelial cell apoptosis produced by UUO, which promoted maintenance of tubular epithelial integrity. OP-1 preserved renal blood flow (RBF) during UUO, but enalapril also stimulated RBF. Thus OP-1 treatment inhibited tubular epithelial disruption stimulated by the renal injury of UUO, preventing tubular atrophy and diminishing the activation of tubulointerstitial inflammation and fibrosis and preserving renal function.

Assessment of the genotoxic potential of ISIS 2302: a phosphorothioate oligodeoxynucleotide.

ISIS 2302, a phosphorothioate oligodeoxynucleotide with antisense activity against human ICAM-1 mRNA, was evaluated in a battery of tests to assess genotoxic potential. There was no evidence of genotoxicity in three in vitro studies performed: (i) a bacterial reverse mutation test; (ii) a chromosomal aberration test in Chinese hamster ovary cells; (iii) a mammalian cell gene mutation assay in L5187Y cells. Additionally, there was no in vivo evidence of genetic toxicity in a bone marrow micronucleus study in male and female mice. For all tests, top concentrations or doses assessed met harmonized regulatory guidelines. The cellular uptake of ISIS 2302 into target cells was confirmed using capillary gel electrophoresis and immunohistochemistry. Intracellular uptake into CHO cells, L5187Y cells, Salmonella typhimurium TA98 and bone marrow was concentration- and time-dependent. Consistent with what is known about the physical and chemical properties of phosphorothioate oligodeoxynucleotides, there was no evidence of genotoxicity in any of the assessed end-points. Furthermore, the absence of genotoxicity could not be ascribed to test system insensitivity or to an absence of exposure of the test system to ISIS 2302.

Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease.

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb(3); also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified alpha-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered alpha-gal A, (ii) to assess the pharmacokinetics of i.v.-administered alpha-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb(3). alpha-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified alpha-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of alpha-gal A, nine of the 10 patients had significantly reduced Gb(3) levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of alpha-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb(3) burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.