Gender differences in serotype 2 adeno-associated virus biodistribution after administration to rodent salivary glands.

Salivary glands (SGs) have proven useful targets for clinical applications of gene therapeutics. In this toxicology and biodistribution study, which conforms to U.S. Food and Drug Administration Good Laboratory Practice regulations, four doses (10(7)-10(10) particles) of a serotype 2 adeno-associated viral (AAV2) vector encoding human erythropoietin were directly administered to the right submandibular gland of male and female BALB/c mice (n = 21 per gender dose group). Control-treated (saline administered; n = 66) and vector-treated (n = 168) animals did not differ in clinical appearance, morbidity and mortality rates, food and water consumption, weight gain ratios, and final weight. Clinical hematology values also were unaffected by AAV2 administration except for parameters influenced by the expression of the recombinant protein (e.g., hematocrit). Mice were killed on days 3, 30, 55, and 92. No major vector-related toxicity was uncovered after complete pathology and histopathology review. However, a significant gender-related difference in vector biodistribution was revealed by quantitative polymerase chain reaction. In male mice vector (group receiving 10(10) particles/animal) effectively transduced, and was primarily confined within, the SGs (i.e., approximately 800 times more copies in SGs than in liver; day 3) and long lived. In contrast, in female mice, SG transduction was less efficient (260-fold less than in males; day 3) and short lived, and vector was disseminated widely via both the bloodstream (SG:liver copy ratio, approximately 1) and saliva (30-fold greater than in males). The observed vector biodistribution is likely due to differences in AAV2 receptor targets and structural differences affecting SG integrity. Sexual dimorphism is a factor of major significance that could potentially affect gene therapy clinical applications in SGs.

Toxicity and biodistribution of a first-generation recombinant adenoviral vector, in the presence of hydroxychloroquine, following retroductal delivery to a single rat submandibular gland.

OBJECTIVE: We examined the toxicity and biodistribution associated with a single administration of a first-generation, serotype 5, adenoviral vector encoding human growth hormone (hGH; AdCMVhGH) to a single rat submandibular gland in the presence of hydroxychloroquine (HCQ). Previously, we showed that hGH is primarily secreted into saliva (approximately ninefold serum level) when expressed as a transgene in salivary glands (e.g. Baum et al, 1999), but administration of HCQ substantially increases the hGH levels secreted into the bloodstream (Hoque et al, 2001). A potential application of this observation is for patients with adult hGH deficiency. METHODS: Six groups of male and female adult rats (n = 12 each) were studied, with zero to 1.5 x 10(11) particles of AdCMVhGH, +/-HCQ, administered retroductally. Multiple clinical and pathological parameters, as well as vector tissue distribution, were assessed. RESULTS: All animals survived until the scheduled day of sacrifice, and essentially no untoward events were observed clinically or at gross necropsy. We observed no vector-related effects on clinical hematology evaluations and a single, transient significant change on clinical chemistry evaluations (increased serum globulin levels). Three days after AdCMVhGH administration, the vector distributed to all tissues analyzed with the exception of gonads and heart. By day 29, most organs, other than the targeted and contralateral submandibular glands, were negative for the presence of vector. On day 3, none of the animals tested positive for the presence of replication competent adenovirus in either their blood or saliva. CONCLUSION: Salivary gland delivery of AdCMVhGH +/-HCQ appears associated with limited toxicity in rats.

Carcinogenicity of dermally administered 1,2-dihydro-2,2,4-trimethylquinoline monomer in F344 rats and B6C3F1 mice.

1,2-Dihydro-2,2,4-trimethylquinoline (TMQ) was evaluated in a 2-year study in which groups of 60 male or female F344 rats received 0, 36 or 60 mg kg(-1) (0, 0.022, or 0.037 mg cm(-2)) and groups of 60 male or female B6C3F1 mice received 0, 3.6 or 10 mg kg(-1) (0, 0.00136, 0.00435 mg cm(-2)) in acetone by topical administration. Survival of all treated groups was comparable to survival of controls. Mean body weights of female rats were lower than those of controls throughout the study but mean body weights of male rats and male and female mice were comparable to the mean body weights of controls. No neoplasms of the skin were observed in any group of rats or mice. Acanthosis at the site of application was increased in male and female rats that received 60 or 100 mg kg(-1) and hyperkeratosis was increased in female rats that received 60 mg kg(-1). The incidences of renal tubule adenoma and renal tubule adenoma or carcinoma were increased significantly in the 60 and 100 mg kg(-1) groups of male rats. There were no neoplastic or non-neoplastic lesions in mice associated with exposure to 1,2-dihydro-2,2,4-trimethylquinoline. In a 1-year initiation-promotion study, groups of 30 female SENCAR mice received an initiating dose of 50 mg kg(-1) 1,2-dihydro-2,2,4-trimethylquinoline followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), or an initiating dose of 7,12-dimethylbenzanthracene (DMBA) followed by promotion with 5, 10 or 25 mg kg(-1) 1,2-dihydro-2,2,4-trimethylquinoline. Other groups served as initiator control, promoter control, vehicle control and positive control (DMBA initiation, TPA promotion). In this system, 1,2-dihydro-2,2,4-trimethylquinoline-initiated skin was not promoted by TPA, and DMBA-initiated skin was not promoted by 1,2-dihydro-2,2,4-trimethylquinoline.

Glycine modulates the toxicity of benzyl acetate in F344 rats.

The influence of supplemental glycine on benzyl acetate (BA; a compound metabolized via the hippurate pathway)-induced toxicity was investigated. Groups of male F344 rats were fed NIH-07 diet containing 0, 20,000, 35,000, or 50,000 ppm BA for up to 28 days. Two additional groups were fed NIH-07 diet with 50,000 ppm BA and 27,000 ppm glycine or 50,000 ppm BA 32,000 ppm L-alanine; supplemental glycine and L-alanine were equimolar. The L-alanine group served as an amino nitrogen control. A third group was fed NIH-07 diet with 32,000 ppm L-alanine and served as an untreated isonitrogenous control BA caused increase in mortality, body weight loss, the incidence of abnormal neurobehavioral signs such as ataxia and convulsions, along with astrocyte hypertrophy and neuronal necrosis in the cerebellum, hippocampus, and pyriform cortex of the brain. These effects were reduced significantly by supplementation with glycine but not with L-alanine. These results suggest that the neurodegeneration induced by BA is mediated by a depletion of the glycine pool and the subsequent excitotoxicity.

Toxicity of furfuryl alcohol to F344 rats and B6C3F1 mice exposed by inhalation.

Groups of F344 rats and B6C3F1 mice were exposed to furfuryl alcohol vapor for 6 hours per day, 5 days per week for 14 days (0, 16, 31, 63, 125, 250 ppm) or 13 weeks (0, 2, 4, 8, 16, 32 ppm). Reduced survival was observed in the 14-day study at 250 ppm. Final mean body weights of rats and mice exposed to 125 ppm and of female mice exposed to 63 ppm were lower than controls at the end of the 14-day study; there were no significant differences in mean body weight among chemical-exposed and control groups in the 13-week study. Exposure to furfuryl alcohol had no toxicologically significant effect on organ weights in either rats or mice, and did not cause any adverse changes in hematology or serum chemistry parameters evaluated in rates in the 13-week study. Microscopic lesions associated with exposure to furfuryl alcohol were present in the nose of both rats and mice at all exposure concentrations in both the 14-day and 13-week studies. Lesions observed in the 14-day study consisted of inflammation of the nasal turbinates accompanied by necrosis and squamous metaplasia of the respiratory epithelium and necrosis and degeneration of the olfactory epithelium. Similar lesions were observed in both rats and mice in the 13-week study. In addition, squamous metaplasia and goblet cell hyperplasia of the respiratory epithelium, squamous metaplasia of the transitional epithelium and degeneration, hyperplasia and some respiratory metaplasia of the olfactory epithelium were also observed in rats in the 13-week study, and hyaline droplets in the respiratory epithelium and chronic inflammation and respiratory metaplasia in the olfactory epithelium were observed in mice in the 13-week study. In general the nasal passages of mice appeared less sensitive than those of rats at the concentrations used in the 13-week study; a no-observable-effect level was not achieved in either the 14-day or the 13-week study.

Carcinogenicity of glycidol in F344 rats and B6C3F1 mice.

Glycidol, a simple aliphatic epoxide, was administered by gavage in water to groups of male and female F344/N rats and B6C3F1 mice. Rats received 0, 37.5 or 75 mg kg-1 and mice received 0, 25 or 50 mg kg-1 daily, 5 days per week for 2 years. Exposure to glycidol was associated with dose-related increases in the incidences of neoplasms in numerous tissues in both rats and mice. Survival of rats that received glycidol was markedly reduced compared to the control because of the early induction of neoplastic disease. In male rats, mesothelioma arising in the tunica vaginalis and frequently metastasizing to the peritoneum were considered the major cause of early death. Early deaths in female rats were associated with mammary gland neoplasms. Survival of female mice that received 50 mg kg-1 was lower than the control after week 101 due primarily to euthanasia of moribund animals with mammary gland neoplasms. Survival of male mice and female mice that received 25 mg kg-1 was comparable to the control. In mice, exposure to glycidol was associated with increased incidences of neoplasms of the harderian gland in males and females, the forestomach in males and the mammary gland in females.

NTP summary report on the metabolism, disposition, and toxicity of 1,4-butanediol (CAS No. 110-63-4).

1,4-Butanediol is an industrial chemical used in the manufacture of other organic chemicals. It was nominated by the National Cancer Institute and selected for evaluation by the NTP because of high production volume, the potential for worker exposure, the lack of adequate toxicological characterization, and the lack of evaluation for carcinogenic potential. As documented in the scientific literature, 1,4-butanediol is rapidly absorbed and metabolized to gamma-hydroxybutyric acid in animals and humans. A metabolism and disposition study conducted in F344/N rats by the NTP confirmed the rapid and extensive conversion of 1-[14C]-1,4-butanediol to 14CO2. Because of this rapid and extensive conversion, the toxicological profile of 1,4-butanediol reflects that of gamma-hydroxybutyric acid. gamma-Hydroxybutyric acid is a naturally occurring chemical found in the brain and peripheral tissues which is converted to succinate and processed through the tricarboxylic acid cycle. Although the function of gamma-hydroxybutyric acid in peripheral tissues is unknown, in the brain and neuronal tissue it is thought to function as a neuromodulator. gamma-Hydroxybutyric acid readily crosses the blood-brain barrier, and oral, intraperitoneal, or intravenous administration elicits characteristic neuropharmacologic responses. These same responses are observed after administration of 1,4-butanediol. The lactone of gamma-hydroxybutyric acid, gamma-butyrolactone, is also rapidly converted to gamma-hydroxybutyric acid by enzymes in the blood and liver of animals and humans. gamma-Butyrolactone was previously evaluated by the NTP in 14-day and 13-week toxicology studies and 2-year toxicology and carcinogenesis studies in F344/N rats and B6C3F1 mice. No organ-specific toxicity occurred in the toxicology studies. In the carcinogenesis studies, an equivocal response occurred in male mice, based on a marginal increase in the incidence of pheochromocytomas of the renal medulla. Because of the rapid and extensive conversion of gamma-butyrolactone to gamma-hydroxybutyric acid, the evaluation of gamma-butyrolactone was in fact an evaluation of gamma-hydroxybutyric acid. This summary report presents a review of the current literature which documents that both 1,4-butanediol and gamma-butyrolactone are rapidly metabolized to gamma-hydroxybutyric acid, and the pharmacologic and toxicologic responses to these chemicals are due to their metabolic conversion to gamma-hydroxybutyric acid. Because the toxicity and carcinogenicity of gamma-hydroxybutyric acid was fully evaluated in the NTP studies of gamma-butyrolactone, and a lack of organ-specific toxicity or carcinogenic potential was demonstrated, it is concluded that there is a high likelihood that 1,4-butanediol would be negative in a similar set of studies. For these reasons, it is the opinion of the NTP that 1,4-butanediol should be considered not carcinogenic in animals and no further evaluation of 1,4-butanediol is needed at this time.

Tumors of the bladder, kidney, and intestine of F344 rats and liver of B6C3F1 mice administered o-nitroanisole in feed.

o-Nitroanisole, a mutagenic intermediate used in the manufacture of azo dyes, was administered in feed for 2 years at concentrations of 0, 222, 666, or 2000 ppm to groups of 60 male and 60 female F344 rats. No significant increase in neoplasms occurred in these groups of rats. Additional (stop exposure) groups of 60 male and 60 female F344 rats received diets containing 0, 6000, or 18,000 ppm for 27 weeks followed by maintenance on control diets for up to an additional 77 weeks. Survival of the stop exposure groups was reduced because of the development of chemical related neoplasms of the urinary bladder. After 13, 28, 40, and 65 weeks on study, 10 rats per group were necropsied and evaluated for the presence of chemical associated lesions. Hyperplasia of the epithelium of the urinary bladder was significantly increased at all interim evaluations. A transitional cell carcinoma was observed at the 13-week evaluation in one male rat that received 18,000 ppm and thereafter transitional cell neoplasms of the bladder were present in male and female rats at each interim evaluation. Adenomatous polyps of the large intestine were significantly increased in groups that received 6000 or 18,000 ppm. In addition carcinomas of the large intestine were present in four males and two females that received 18,000 ppm. Hyperplasia of the transitional epithelium of the renal pelvis was significantly increased in groups of rats that received 6000 or 18,000 ppm and transitional cell papillomas were observed in three males and one female that received 18,000 ppm. Transitional cell carcinomas of the kidney occurred in one male that received 6000 ppm and six males and one female that received 18,000. Groups of 60 male and 60 female B6C3F1 mice received dietary concentrations of 0, 666, 2000, or 6000 ppm o-nitroanisole for 2 years. No stop exposure study was conducted with mice. The only neoplastic response observed in mice was in the liver of males; hepatocellular adenomas or carcinomas were increased in groups of male mice that received 2000 or 6000 ppm. No increase in neoplasms associated with chemical exposure occurred in female mice.

Induction of thymic lymphoma in mice administered the dideoxynucleoside ddC.

Groups of 10 male and 20 female B6C3F1 mice received 0, 500, or 1000 mg/kg/day 2'3'-dideoxycytidine (ddC) by gavage for 13 weeks. At the end of the 13-week exposure period all males and 10 females per group were necropsied while the remaining females were held for 1 month without further treatment. Thymic atrophy was present at the 13-week necropsy in male and female mice administered 1000 mg/kg/day and in females administered 500 mg/kg/day, but was not present in females following 1 month of recovery. Thymic lymphoma was present in 1 female that received 500 mg/kg/day and 1 female that received 1000 mg/kg/day. In a follow-up study groups of 70 female mice received 0, 500, or 1000 mg/kg/day for 13 weeks. At the end of the 13-week exposure period 20 mice per group were necropsied and the remaining animals held for 3 months without further treatment. Thymic atrophy was observed in ddC-exposed groups at the 13-week necropsy but not in mice allowed to recover for 13 weeks. Thymic lymphoma occurred in 3/50 mice that received 500 mg/kg/day and in 17/50 mice that received 1000 mg/kg/day but did not occur in mice from the vehicle control group.

Mixed dentition analysis: a review of methods and their accuracy.

Methods that have been proposed for mixed dentition analysis are reviewed. Prediction of the space required in the dental arch for unerupted permanent canines and premolars has been based either on the correlation between the mesio-distal widths of these teeth and of erupted mandibular incisors, or on measurements of the unerupted teeth on radiographs. Studies comparing the different methods have shown that the method of Hixon & Oldfather (1958), as refined by Staley & Kerber (1980), is the most accurate.

1,2,3-Trichloropropane: a multisite carcinogen in rats and mice.

1,2,3-Trichloropropane was evaluated in 2-year toxicology and carcinogenesis studies by the National Toxicology Program. The selection of this chemical for study was based on the potential for human exposure, its positive in vitro genotoxicity, and the carcinogenicity of structurally related chemicals. During the 2-year study 1,2,3-trichloropropane was administered in corn oil by gavage 5 days per week; groups of 60 F344/N rats received 0, 3, 10, or 30 mg/kg, while groups of 60 B6C3F1 mice received 0,6,20, or 60 mg/kg. Because of reduced survival associated with the development of chemical-related neoplasms, rats that received 30 mg/kg were terminated at 65 weeks (females) or 76 weeks (males). Similarly, mice that received 60 mg/kg were terminated at 73 weeks (females) or 79 weeks (males), while groups of mice that received 20 mg/kg were terminated at 88 weeks. 1,2,3-Trichloropropane induced benign and/or malignant neoplasms at multiple sites in both rats and mice; this included increased incidences of benign and malignant neoplasms of the squamous epithelium of the oral mucosa and forestomach of male and female rats, benign neoplasms of the kidney and pancreas and benign or malignant neoplasms of the preputial gland in male rats, malignant neoplasms of the mammary gland, and benign or malignant neoplasms of the clitoral gland in female rats. In mice, 1,2,3-trichloropropane induced a low incidence of malignant neoplasms of the oral mucosa in females, high incidences of benign and malignant neoplasms of the forestomach in males and females, benign neoplasms of the liver and harderian gland of males and females, and uterine neoplasms in females.

Toxicokinetics of pentachlorophenol in the F344 rat. Gavage and dosed feed studies.

1. The toxicokinetics of pentachlorophenol (PCP) were studied in the Fischer 344 rat using i.v. and oral (gavage, dosed feed) routes of exposure. 2. Only minor sex differences were observed in the elimination kinetics of PCP after i.v. administration at 5 mg/kg. 3. Absorption of PCP from the gastrointestinal tract after gavage doses of 9.5 and 38 mg/kg in aqueous methylcellulose vehicles was first order with an absorption half-life of about 1.3 h. 4. The absorption rate constant of PCP from doses feed was comparable with that obtained from aqueous methylcellulose gavage formulations. 5. Bioavailability of PCP administered in dosed feed was significantly lower than the bioavailability of PCP administered by gavage. 6. Dose proportionality was established to a dosage of at least 38 mg/kg. 7. Daily fluctuation of PCP plasma concentrations was observed during the dosed feed study with peak and trough concentrations occurring in early morning and late afternoon, respectively. 8. The time course of PCP plasma concentrations during the dosed feed study were simulated using a computer model based on linear theory. The simulations were comparable with the experimentally determined concentrations.

Zidovudine bioavailability and linear pharmacokinetics in female B6C3F1 mice.

The bioavailability and pharmacokinetics of zidovudine (3'-azido-3'-deoxythymidine) (AZT) were determined in female B6C3F1 mice after administration of 15, 30, and 60 mg/kg doses via oral gavage or intravenous injection. Three animals in each administration group were sacrificed, and blood samples were collected at each of the following times: 0, 5, 10, 15, 20, 30, 45, 60, 75, 90, 105, and 120 min after drug administration. Plasma zidovudine concentrations were determined by HPLC. After oral administration, mean maximum plasma concentrations (Cmax) of 9.1, 18.9, and 40.3 mg/liter were observed at 18.3, 21.7, and 15.0 min (tmax) for the 15, 30, and 60 mg/kg doses, respectively. Following intravenous administration, mean Cmax values of 15.9, 41.8, and 76.0 mg/liter were observed for the 15, 30, and 60 mg/kg doses, respectively. Nonlinear least squares regression of all data sets, using a 1/y weight, indicated that zidovudine disposition was best described by a one-compartment open model with first-order absorption, where appropriate, and first-order elimination. The mean elimination half-life values ranged from 17.3 to 19.9 min for the three intravenous doses and from 16.5 to 21.9 min for the three oral doses. The mean values for the apparent volume of distribution (Vd) ranged from 0.8 to 1.0 liter/kg following oral and intravenous administration. There were no significant differences in Vd between the oral and intravenous groups. The mean total body clearance values ranged from 28.9 to 34.3 ml/min/kg following intravenous administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of [14C]furan in the male F344 rat.

In a recently completed 2-yr bioassay, furan was found to induce cholangiocarcinomas at high incidence in rats. The disposition of single and multiple gavage doses of [2,5-14C]furan has been determined in male F344 rats to aid in interpretation of that study. In the 24 h after dosing about 80% of the furan-derived radioactivity was eliminated, primarily via urine and expired air. [14C]Carbon dioxide was a major metabolite, indicating that furan ring opening followed by complete oxidation of at least one of the labeled carbons was a major part of the overall metabolism of furan. Liver contained more furan-derived radioactivity by far than other tissues after 24 h. Approximately 80% of the radioactivity in liver was not extracted by organic solvents and was associated with protein. There was either no binding to DNA or the furan-DNA adduct was not stable to the isolation procedure. Repeated daily administration of [14C]furan resulted in a more or less linear increase in covalent binding through four doses; at this point the amount of nonextractable radioactivity plateaus. Urine contained at least 10 metabolites, again indicating extensive metabolism of the furan ring. From the data obtained in this study it is clear that furan is metabolized to reactive species, apparently primarily in liver, and these intermediates react with protein. The hepatotoxicity resulting from furan exposure may be due to the reaction of furan metabolites with liver macromolecules; the presence of some of these reactive metabolites following chronic exposure to furan may result in cholangiocarcinomas.

Hematologic toxicity of AZT and ddC administered as single agents and in combination to rats and mice.

Toxicity studies of 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) were conducted in F344/N rats and B6C3F1 mice. The drugs were administered as single agents and in combination. In all studies, animals were treated by oral gavage twice a day, 7 days a week. In studies of the individual compounds, each was administered for 13 weeks at the following concentrations: AZT in rats, 0, 125, 250, 500, 1000 mg/kg and in mice, 0, 25, 50, 100, 400, 1000 mg/kg; ddC in rats and mice, 0, 250, 1000, 2000 mg/kg. Additional male rats and female mice that were treated with 0, 250, 1000, or 2000 mg/kg ddC and male and female mice treated with 0, 50, 400, 1000 mg/kg AZT were maintained for 30 days after treatment was stopped (at 94 days) to evaluate the reversibility of toxic effects. Hematologic variables were measured on Days 5, 23, and 94 (last day of dosing), and on Day 123 (after a 30-day period without treatment). AZT and ddC produced dose-related, poorly regenerative, macrocytic anemias as evidenced by decreases in erythrocyte counts, hematocrits, and hemoglobin concentrations and increases in mean corpuscular hemoglobin and mean corpuscular volume. Bone marrow samples in rats treated with AZT were hyperplastic whereas those in mice treated with AZT and rats and mice treated with ddC were hypoplastic. The hematologic toxicity of AZT was more severe than that of ddC. Generally, toxic effects of either chemical were greater in mice than in rats and more pronounced in female than in male animals. After 30 days without dosing, hematologic effects either resolved or dramatically improved. In studies in which ddC and AZT were administered in combination for 4 weeks at concentrations of 0/0, 0/500, 500/0, 10/500, 100/500, 500/500, and 500/1000 mg/kg ddC/AZT, there was macrocytic anemia in animals in the lower doses and marked microcytic anemia in surviving male mice in higher dose groups. Most female mice died in the 500/500 and 500/1000 mg/kg ddC/AZT dose groups. At lower concentrations (100/500, 500/1000 mg/kg ddC/AZT), effects of the two drugs were similar to those in the single drug studies. At higher concentrations (500/500 and 500/1000 mg/kg ddC/AZT), the combination treatment produced enhanced hematopoietic toxicity. These studies demonstrated the early and progressive time course of toxicity of AZT and ddC, species differences in sensitivities and responses, and reversibility of effects after termination of treatment. Based on these findings, a chronic toxicity study is being conducted with AZT in mice.

Furan-induced hepatic cholangiocarcinomas in Fischer 344 rats.

In a 2-yr carcinogenicity bioassay, 0, 2, 4, or 8 mg furan/kg body weight (BW) was administered to male and female Fischer (F344) rats and resulted in an 86-100% incidence of cholangiocarcinomas with occasional metastasis. In a separate but concurrent study, male F344 rats dosed with 30 mg furan/kg BW for 90 days developed marked cholangiofibrosis and cholangiohepatitis and, when subsequently maintained without further treatment for an additional 6, 12, or 18 months, the cholangiofibrosis progressed to yield a 100% incidence of cholangiocarcinomas. Transplantation of 21 primary cholangiocarcinomas into syngeneic recipients resulted in growth from 4 donors. The 4 transplanted lines were successfully transferred through 8 serial passages and resulted in metastases in the recipients. The progressive growth of these proliferative hepatocholangial lesions over time, their transplantability, and the development of metastases in some of the cases provide biological evidence of the malignant potential of the furan-induced liver changes.

Cross-linking of neurofilament proteins of rat spinal cord in vivo after administration of 2,5-hexanedione.

The aliphatic hexacarbons n-hexane, methyl-n-butyl ketone, and 2,5-hexanedione are known to produce a peripheral neuropathy that involves an accumulation of 10-nm neurofilaments above the nodes of Ranvier in the spinal cord and peripheral nerve. In this study, rats were treated with 0.5% 2,5-hexanedione in drinking water for 180 days, and their spinal cord neurofilaments were isolated after development of the neuropathy. Visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a significant reduction in content of the neurofilament triplet proteins in treated animals and the presence of bands migrating at 138K and 260K that were not present in control animals. Analysis of the lanes using immunoblotting procedures and anti-70K, anti-160K, and anti-210K neurofilament antibodies revealed many cross-linked peptides. The 138K band cross-reacted with the anti-160K neurofilament antibody. This suggests that the 138K band is an intramolecular cross-link of the 160K neurofilament subunit. In addition to this peptide, there were numerous high-molecular-weight peptides immunoreactive with all three neurofilament protein antibodies. In addition to cross-linking, there was also a diminished amount of immunoreactive breakdown product of all three neurofilament proteins. This report demonstrates direct evidence of 2,5-hexanedione-induced cross-linking of neurofilament proteins in vivo, which maybe responsible for the accumulation of neurofilament proteins pathognomic of this neuropathy.

The effect of 2450-MHz microwave radiation during microtubular polymerization in vitro.

Exposure to 2450-MHz (cw) microwave radiation causes inhibition of cell division in intact cells and varied in vivo biological effects in both avian and mammalian species. Because these reported effects may result from alterations in the dynamics of microtubule formation, we studied the effects of simultaneous microwave exposure (2450 MHz, cw) during each of the three critical stages of the intracellar polymerization cycle. In addition, using circular dichroism spectroscopy, we studied the effect of microwave irradiation on the secondary structure of purified tubulin polypeptides. These studies were accomplished using specially constructed exposure systems that permit the continuous recording of turbidometric or circular dichroism measurements during simultaneous exposure to microwaves. The baseline turbidity of microtubular protein did not change under the influence of microwave radiation (20 or 200 mW/g SAR) and irradiation had no effect on the light-scattering properties of the depolymerized protein. EGTA-induced polymerization and cold-induced depolymerization patterns were also similar for both control and microwave-irradiated samples. The circular dichroism spectrum of purified tubulin also did not appear to be influenced by microwave irradiation, indicating a lack of effect on the protein secondary structure. The data suggest that the cellular effects of microwaves are not due to changes in microtubular proteins or their rate of polymerization.

Effects of ionizing radiation on the polymerization of microtubules in vitro.

Exposure of phosphocellulose-purified 6S tubulin to ionizing radiation results in a reduction or loss in its ability to participate in polymerization. Evidence is presented which correlates this loss in ability to polymerize with a reduction in the number of titratable sulfhydryl groups and a lowered affinity for guanosine triphosphate.