A 2-year comparison study of Crl:CD BR and Hsd:Sprague-Dawley SD rats.

In this 2-year study, the suitability of the Hsd:Sprague-Dawley SD (SD) as a replacement for the Crl:CD BR (CD) rat was assessed by comparing survival rates, palpable mass incidence, body weights, food consumption, clinical laboratory parameters, and necropsy and histopathology observations. At week 104, survival rates in the CD and SD males were 29 and 49%, respectively. Corresponding survival rates in females were 44 and 63%. The total numbers of animals with palpable masses and animals with neoplasms were similar in the CD and SD rats; however, the total numbers of palpable masses and neoplasms were higher in the CD rats. The incidence of corneal lesions was higher in the SD rats, whereas the incidence of lenticular opacities was higher in the CD rats. Body weights, food and water consumption, and organ weights were significantly lower in the SD rats. In contrast, food intake per kilogram of body weight was slightly higher in the SD rats. Numerous differences in clinical laboratory parameters between the CD and SD rats were observed. Some of these were consistent with the increased prevalence of kidney disease and secondary sequelae in the SD rats. Taken together, the better survival, smaller size, and lower food consumption of the SD rat may make it a better model for chronic bioassays. However, the increased propensity for spontaneous renal disease may limit the utility of the SD rat for studying nephrotoxic compounds.

Correlation of frameshift mutagenicity with DNA intercalation by CGS 20928A using an in vitro DNA unwinding assay.

A compound's mutagenicity in different Salmonella tester strains can suggest its mechanism of reaction with DNA. Clear confirmation of such a mechanism, however, requires a direct test of the compound's reaction with DNA, often relying on specific in vitro studies. We report the use of a rapid in vitro test designed to measure DNA unwinding, a characteristic of DNA intercalators and many frameshift mutagens. CGS 20928A, an adenosine antagonist, produced a significant (> 2-fold) increase in revertants only for Salmonella tester strain TA1537, and only without metabolic activation. These data indicated that the compound was a direct acting frameshift mutagen and possibly intercalated into DNA. Our DNA unwinding assay indicated that at concentrations of > 0.1 mM CGS 20928A behaved like known intercalating compounds in that it unwound DNA. These concentrations of compound are comparable to those found mutagenic to TA1537. By comparison, the frameshift mutagen and known intercalating compound 9-aminoacridine unwound DNA in this assay in a concentration dependent fashion between 6-12 microM. ICR-191, another acridine frameshift mutagen, also unwound DNA. A compound structurally related to CGS 20928A, which was not mutagenic in Salmonella tester strains, did not produce any DNA unwinding even at 10 mM. Because the assay uses microgram quantities of material, it should be ideal for screening small amounts of congeneric series suspected of frameshift mutagenicity.

Development of an optimal S9 activation mixture for the L5178Y TK+/- mouse lymphoma mutation assay.

In previously described activation systems [Clive D, Spector JFS (1975): Mutat Res 31:17-29] for the mouse lymphoma mutation assay the cofactor isocitrate is rapidly exhausted and the resultant loss of NADPH can halt metabolic processes. Presented here are data obtained with a non-toxic balance of NADP (1.4 mg/ml), isocitrate (6.0 mg/ml), and S9 (less than or equal to 4%) in Fischer's medium which produces a more stable supply of the required cofactors. By spectrophotometric analysis, the molar concentration of NADPH remains at greater than or equal to 50% or more of the maximum over the usual 4-hr treatment period. Accompanying this increase in NADPH duration was increased toxicity and mutant frequency at most doses among cells treated with the reference mutagens 3-methylcholanthrene (MCA), 2-acetylaminofluorene (AAF), benzo(a)pyrene (BAP), 9,10-dimethyl-1,2-benzanthracene (DMBA), or cyclophosphamide (CPA), but not with dimethylnitrosamine (DMN)-possibly a reflection of the single enzyme mediated step in the metabolism of this chemical. These observations also suggest that results attributed to varying the amounts of S9 in an activation mixture may be due to suboptimal cofactor levels and further emphasize the need to maintain sufficient NADPH exposure to evaluate the effects of metabolic enzyme levels or compare the relative activities of analogous chemicals.

GC-rich regions in genomes as targets for DNA alkylation.

For many DNA-damaging agents, the extent of damage at any given base site is influenced by the DNA sequence surrounding that site. Most agents that alkylate the guanine N7 position, including mechlorethamine (nitrogen mustard) and benzo[a]pyrene diol epoxide, alkylate oligo-guanine sequences preferentially. Since these data suggest that guanine-cytosine(GC)-rich regions in genes could be preferred sites of damage by these agents, GenBank was searched for genes containing 30 bp sequences of greater than 90% GC (GC runs). While primate, rodent, other mammalian, vertebrate and animal virus genes constituted 57% of the annotated entries, they included 90% of the entries with the GC runs. In addition, the percentage of oncogenes in the group of the entries with GC runs was higher than that in the overall database. One gene of interest containing GC runs was the human c-Ha-ras oncogene. All seven GC runs in the c-Ha-ras gene are in the 5'-flanking region, rather than in the coding sequences. In fact, some of the GC runs are contained in Sp1-binding enhancer sequences. Gel analysis of the alkylation of cloned c-Ha-ras DNA by several carcinogenic alkylating agents strongly suggest that in this gene GC runs can be preferred sites of damage. These observations suggest mechanisms by which DNA damage at sites other than oncogene coding sequences may play a role in carcinogenesis and/or chemotherapy.

Mechanisms of neurotoxicity related to selective disruption of microtubules and intermediate filaments.

The neuronal response to several neurotoxic chemicals includes disruption of the cytoskeleton such as interactions with microtubules and altered distribution of neurofilaments. Methylmercury (microtubule disrupting) and acrylamide and 2,5-hexanedione (neurofilament disrupting) have been used in a cell culture (PtK2) system to distinguish the cytoskeletal targets of these compounds. Methylmercury caused disassembly of microtubules with secondary collapse of vimentin filaments (epithelial cell equivalent of neurofilaments) at higher concentrations; actin filaments were unaltered. This confirms that disruption of actin does not contribute to methylmercury-induced interference with mitosis. In contrast, both acrylamide and 2,5-hexanedione caused a perinuclear redistribution of vimentin filaments with sparing of microtubules. Biochemical studies revealed that 2,5-hexanedione treatment resulted in high molecular weight vimentin-immunoreactive species, presumably by cross-linking of proteins. Selective action of both acrylamide and 2,5-hexanedione on vimentin filaments and the similarity of effects suggest that a common mechanism of damage may occur whereby these compounds act directly on both vimentin and neurofilaments.

Specific and nonspecific effects of EMG biofeedback.

This study investigated the effects of performance feedback and EMG biofeedback on perceptions of the "self" (i.e., self-esteem, self-control, self-efficacy, and locus of control) as well as on a self-control behavior (study skills) the subjects performed outside the laboratory. Forty-seven college students were randomly assigned to one of four groups in a 2(high and low success feedback) x 2(true and false EMG biofeedback) factorial experiment with repeated measures. All of the participants received four sessions of EMG biofeedback, and later they were asked to self-monitor their study habits for 2 weeks. Results showed that the self-esteem measure and perceptions of study skills improvement were differentially affected by success feedback but unrelated to the true or false EMG manipulation. Shifts toward an internal locus of control and perceptions of improved self-control were also noted, but they were independent of the subjects' group membership. Implication of the results are briefly discussed.

Nick translation--a new assay for monitoring DNA damage and repair in cultured human fibroblasts.

An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. This nick translation assay, originally proposed by Nose and Okamoto [1983], is very sensitive, allows detection and quantitation of both DNA damage and repair, distinguishes between various types of induced damage, does not require radioactive prelabeling of cells, and circumvents some of the problems inherent in unscheduled DNA synthesis (UDS) assays. The assay is also useful in detecting those agents that inhibit replicative DNA synthesis and/or the excision repair process. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

Vibration effects on three measures of relaxation.

The effect of vibrotactile stimulation on relaxation as measured by EMG recording of the frontalis and trapezius muscles and by subjective report was assessed. It was predicted that low-frequency vibrotactile stimulation (less than 70 Hz) would facilitate muscle relaxation when measured by both EMG frontalis and trapezius recordings and by subjective report. The participants (8 male and 8 female) were randomly assigned to split-plot, before/after design consisting of four between-subjects treatments and one within-subjects treatment (pre- and post-treatment). The between-subjects treatments were footrest vibration, backrest vibration, footrest-backrest vibration combined, and control. The within-subjects treatment included pre- and post-treatment levels. Results of repeated-measures analyses of variance on each set of data yielded a significant change from pre- to posttreatment condition on all EMG and subjective report measures of muscle tension except the control. The utility of using EMG as a measure of relaxation is discussed.

Lack of cytogenetic effects in mice or mutations in Salmonella receiving sodium fluoride.

We have examined the possible effect of fluoride intake on chromosome damage. There was no evidence of increased frequency of chromosomal aberration in bone marrow or testis cells of mice with either 50 ppm fluoride intake over several generations or 100 ppm intake for 6 weeks compared to animals drinking distilled water. Fluoride was not found to be mutagenic in a widely used bacterial mutagenesis assay over a range of 0.1 to as high as 2000 microgram fluoride per plate.

Erythrocyte shape in Duchenne muscular dystrophy.

In a controlled and blind study we were not able to identify any abnormality of erythrocytes of eight patients with Duchenne muscular dystrophy compared with seven approximately age-matched unaffected siblings (six males, one female step-sibling). Erythrocyte morphology was found to be very sensitive to various types of cell treatment. At this time, erythrocyte morphology, evaluated by the techniques used, should not be considered an established diagnostic test for the Duchenne muscular dystrophy patient or carrier.

Relaxation measured by EMG as a function of vibrotactile stimulation.

The present study investigated the effect of vibrotactile stimulation on relaxation as measured by EMG recording. Forty-eight subjects from three age groups were randomly divided into 8 experimental groups: (1) simultaneous footrest vibration and back vibration (A1C1); (2) simultaneous footrest vibration and back roller (A1C2); (3) simultaneous footrest vibration, back vibration and back roller (A1C3); (4) footrest vibration alone (A1C4): (5) back vibration alone (A2C1); (6) back roller alone (A2C2); (7) simultaneous back vibration and back roller (A2C3); and (8) control group (no vibration/stimulation) (A2C4). The three major variables studied were footrest vibration (A1 and A2), pre- and post-EMG measures (B1 and B2), and back vibration (C1C2C3C4). Results showed that footrest vibration had a significant effect on relaxation. Other conditions (except the control) produced a decrease in EMG levels, but did not reach significance. Pre- and postmeasures by experimental conditions were also significantly different. Application of vibration as an aid in relaxation is discussed.

Erythrocyte deformation in human muscular dystrophy.

Erythrocytes from patients with congenital muscular dystrophy exhibit dramatic surface deformation when observed with a scanning electron microscope. A similar alteration, but one affecting a smaller proportion of cells, occurs in the case of female carriers of the sex-linked Duchenne dystrophic condition. These observed changes in the erythrocyte surface may reflect a systemic defect in membrane properties.