Reaction of the antitumor antibiotic CC-1065 with DNA: structure of a DNA adduct with DNA sequence specificity.

Sequence-dependent variations in DNA revealed by x-ray crystallographic studies have suggested that certain DNA-reactive drugs may react preferentially with defined sequences in DNA. Drugs that wind around the helix and reside within one of the grooves of DNA have perhaps the greatest chance of recognizing sequence-dependent features of DNA. The antitumor antibiotic CC-1065 covalently binds through N-3 of adenine and resides within the minor groove of DNA. This drug overlaps with five base pairs for which a high sequence specificity exists.

Tick bite and Lyme disease-related emergency department encounters in New Hampshire, 2010-2014.

Lyme disease (LD) is a common tick-borne disease in New Hampshire (NH). While LD is a reportable condition and cases are counted for public health surveillance, many more people receive care for tick bites or diagnoses of LD than are reflected in surveillance data. NH's emergency department (ED) data system was queried for tick bite and LD-related encounters. Chief complaint text was queried for words related to LD or tick bites. International Classification of Diseases 9th Revision (ICD-9) codes were queried for the LD diagnosis code (088.81). Emergency department patient data were matched to reportable disease data to determine the proportion of ED patients reported to the health department as a suspected LD case. Data were analysed to calculate frequencies for key demographic and reporting characteristics. From 2010 to 2014, 13,615 tick bite or LD-related ED encounters were identified in NH, with most due to tick bites (76%). Of 3,256 patients with a LD-related ED encounter, 738 (23%) were reported to the health department as a suspected LD case. The geographic distribution of ED patients was similar to reported LD cases; however, the regions of the state that experienced higher rates of ED encounters were different than the regions that observed higher rates of reported LD cases. Seasonal distribution of ED encounters peaked earlier than reported LD cases with a second peak in the fall. While age and sex distribution was similar among ED patients and reported LD cases, the rates for children 5 years and younger and adults 65 years and older were greater for ED encounters. Patients frequently visit the ED to seek care for tick bites and suspected LD. Results of ED data analyses can be used to target education, in particular for ED providers and the public through timely distribution of evidence-based educational materials and training programmes.

Synthesis of 1-(2-hydroxyethyl)-1-nitrosourea and comparison of its carcinogenicity with that of 1-ethyl-1-nitrosourea.

1-(2-Hydroxyethyl)-1-nitrosourea (HNU) was prepared by the action of nitrosyl chloride on (2-hydroxyethyl)urea. Attempts to synthesize HNU by an earlier described method were unsuccessful and led to the formation of the cyclized derivative 1-nitroso-2-oxazolidone. In addition, the spectral data that we obtained for HNU differed from those reported earlier. Female C57BL/Cbl mice were treated with single ip doses of HNU to determine its median lethal dose (LD50) and its ability to induce lymphocytic thymic lymphomas in these mice. The results showed that the LD50 was the same as that for 1-ethyl-1-nitrosourea (ENU) and that its was slightly more potent than ENU as a carcinogen in this system.

The reactivity and carcinogenicity of aflatoxin B1-2,3-dichloride, a model for the putative 2,3-oxide metabolite of aflatoxin B1.

Aflatoxin B1-2,3-dichloride (AFB1-Cl2) was synthesized as a model for the probable ultimate carcinogen, aflatoxin B1-2,3-oxide. As expected for aflatoxin B1-2,3-oxide, AFB1-Cl2 has an electrophilic carbon 2; it decomposed in water (half-life of 0.5 min in 10% dimethyl sulfoxide, pH 7.4) with the formation of 3-chloro-2,3-dihydro-2-hydroxyaflatoxin B1 and 2,3-dihydro-2,3-dihydroxyaflatoxin B1. AFB1-Cl2 formed covalent adducts with DNA and RNA with retention of one-half of the chlorine; the major products apparently contained glycosidic bonds between carbon 2 of the aflatoxin residues and nitrogen or oxygen atoms in the nucleic acids. Polyguanylic acid was the most reactive homopolymer toward AFB1-Cl2. AFB1-Cl2 was less reactive toward mononucleotides than toward polynucleotides. The major adducts formed on incubation of AFB1-Cl2 with protein contained little chlorine and could have resulted from alkylation of primary amino groups or from reactions with the hydrolysis products. Similarly, incubation of AFB1-Cl2 with amino acids apparently resulted in Schiff base formation between primary amino groups and the dialdehyde rearrangement forms of the hydrolysis products of AFB1-Cl2. AFB1-Cl2 was much more active than aflatoxin B1 in inducing sarcomas at the s.c. injection site in rats, in the initiation of papillomas on the skin of mice, and in the induction of lung tumors in mice. AFB1-Cl2 was also highly mutagenic for Salmonella typhimurium TA 98 and TA 100. Aflatoxin B1 and its 2,3,-dihydro- (aflatoxin B2), 2,3-dihydro-2-hydroxy- (aflatoxin B2a), 2,3-dihydro-2,3-dihydroxy-, and 3-chloro-2,3-dihydro-2-hydroxy- derivatives were inactive in the mutagenicity tests; and the latter four compounds were also inactive as initiators of papillomas of the skin in mice. The structures of the macromolecular adducts of AFB1-Cl2 formed in vitro, the carcinogenicity of this electrophile, and the lack of carcinogenicity of its hydrolysis products indicate that alkylation of nucleic acids is a critical reaction in tumor induction with this carcinogen and aflatoxin B1.

Aflatoxin B1-2,3-oxide as a probable intermediate in the covalent binding of aflatoxins B1 and B2 to rat liver DNA and ribosomal RNA in vivo.

Administration of[3H]aflatoxin B2 (2,3-dihydroaflatoxin B1)(AFB2) to male rats resulted in levels of hepatic DNA- and ribosomal(r)RNA-aflatoxin adducts that were about 1% of those for rats given [3H]aflatoxin B1(AFB1). The levels of hepatic protein-aflatoxin adducts were 35 to 70% as great for AFB2-treated as compared to AFB1-treated rats...

CC-1065 (NSC 298223), a novel antitumor agent that interacts strongly with double-stranded DNA.

CC-1065, a novel antibiotic produced by Streptomyces zelensis, was active against several experimental tumors in vivo and a broad spectrum of human tumor cells in vitro. This report describes its biological and biochemical effects of L1210 leukemia cells. CC-1065 is one of the most cytotoxic agents known. The concentrations required for a 50 and 90% inhibition of cell growth are 0.02 and 0.05 ng/ml, respectively. It is about 400 times more cytotoxic than was Adriamycin. The action of CC-1065 is rapid and is dose and time dependent. CC-1065 inhibits DNA synthesis much more than it inhibits RNA and protein synthesis. The concentrations required for a 50% inhibition of DNA synthesis and RNA synthesis are 4 to 6 and 45 to 60 ng/ml, respectively. Although the drug inhibition of DNA synthesis cannot completely account for its cytotoxic effects on L1210 cells, these results, along with those generated by other investigators, suggest that the inhibition of DNA synthesis represents a major mode of action of CC-1065. CC-1065 inhibited both thymidine kinase and DNA polymerase alpha activities, but the effect on highly purified DNA polymerase alpha was more pronounced. At 1 microgram/ml, CC-1065 inhibited more than 70% of the enzyme activity. In order to elucidate the mechanism of inhibition of DNA polymerase alpha, the interaction between CC-1065 and DNA was investigated. The studies with thermal melting of DNA and difference circular dichroism measurement indicate that CC-1065 is one of the strongest DNA-binding agents. It induced an increase in thermal melting temperature of calf thymus DNA by at least 31 degrees. The circular dichroism studies also reveal that CC-1065 binds only to double-stranded DNA but not to heat-denatured DNA or yeast RNA. These observations were supported by those obtained with two other experimental approaches. CC-1065 also appeared to interact with proteins, but the interaction was weak and reversible.

Mechanism of interaction of CC-1065 (NSC 298223) with DNA.

CC-1065 (NSC 298223), a potent new antitumor antibiotic produced by Streptomyces zelensis, interacts strongly with double-stranded DNA and appears to exert its cytotoxic effects through disruption of DNA synthesis. We undertook this study to elucidate the sites and mechanisms of CC-1065 interaction with DNA. The binding of CC-1065 to synthetic and native DNA was examined by differential circular dichroism or by Sephadex chromatography with photometric detection. The binding of CC-1065 with calf thymus DNA was rapid, being complete within 2 hr, and saturated at 1 drug per 7 to 11 base pairs. The interaction of CC-1065 with synthetic DNA polymers indicated a specificity for adenine- and thymine-rich sites. Agarose gel electrophoresis of CC-1065-treated supercoiled DNA showed that CC-1065 did not intercalate. Site exclusion studies using substitutions in the DNA grooves showed CC-1065 to bind primarily in the minor groove. CC-1065 did not cause DNA breaks; it inhibited susceptibility of DNA to nuclease S1 digestion. It raised the thermal melting temperature of DNA, and it inhibited the ethidium-induced unwinding of DNA. Thus, in contrast to many antitumor agents, CC-1065 stabilized the DNA helix. DNA helix overstabilization may be relevant to the mechanism of action of CC-1065.

Pneumonia in a long-term care facility. A prospective study of outcome.

BACKGROUND: Pneumonia is a major cause of morbidity and mortality among patients in long-term care facilities. OBJECTIVES: To conduct a prospective study of 108 consecutive patients who acquired pneumonia in a Veterans Affairs facility from January through December 1993, and to identify (1) the short- and long-term outcome of pneumonia, (2) the determinants of outcome, and (3) the frequency of recurrent episodes. METHODS: Patient characteristics, including scores from the Activities of Daily Living (ADL) Index of Katz et al and the Comorbidity Index of Charlson et al, were recorded End points were survival at 14 days and 12 and 24 months, recurrent episodes of pneumonia, and hospitalization for nonpneumonic illness. RESULTS: Fourteen-day mortality was 19%; outcome was significantly related to the ADL score. There was no relationship between short-term outcome and age or the Comorbidity Index score. Mortalities at 12 and 24 months were 59% and 75%, respectively. Long-term survival also correlated with the ADL score. For the least debilitated patients (ie, those with an ADL score < or = 10), mortalities were 33% and 48% at 12 and 24 months, respectively; for those with ADL scores of 11 to 15, the corresponding mortalities were 60% and 75%; and for those with ADL scores of 16 or greater, the mortalities were 65% and 77% (P = .02). Within 12 months, 43% of the survivors had additional episodes, and 37% required transfer to an acute care facility for other diagnoses. Functional status did not change among the most dependent patients. CONCLUSIONS: Functional status is the major determinant of survival following pneumonia. Pneumonia in a debilitated patient in a long-term care facility predicts recurrent pneumonia and death within 1 to 2 years.

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Restriction fragment length polymorphisms associated with abnormal lipid levels in an adolescent population.

The association of restriction fragment length polymorphism genetic markers at the apolipoprotein AI-CIII-AIV gene locus with lipid and lipoprotein levels was evaluated in subsets of the 666 Caucasian students from the junior high school (11-14 years old) population of Muscatine, Iowa. Male students whose leukocyte DNA had an uncommon haplotype consisting of the minor allele of a SacI restriction site polymorphism (S2) in combination with the more common allele of an MspI site variation (M1) had significantly lower levels of high density lipoprotein (HDL)-cholesterol (P less than 0.05) when compared to a random sample of males; these levels were stable over a 2-year follow-up. The minor allele of an XmnI restriction site polymorphism (X2) was more frequent in females with triglyceride levels in the upper decile of the age and gender-specific triglyceride distribution than in normotriglyceridemic females (0.10 less than P less than 0.15) and triglyceride levels were higher in random sample females with an X2 allele than in X1 homozygotes (P less than 0.10). These results suggest that alleles of the known apolipoprotein genes (AI, CIII, AIV) at this locus or closely linked gene sequences may have major effects on lipid levels in members of the general population.

Synthesis and evaluation of a boronated nitroimidazole for boron neutron capture therapy.

We postulated that nitroimidazoles, previously used for radiosensitizing solid tumors, may be interesting templates as carriers of 10B for boron neutron capture therapy. To test this hypothesis, we synthesized a 10B-enriched nitroimidazole, 1-2[(undecahydro-closo-dodecaborato)thio]ethyl]-2- methyl-5-nitroimidazole (imidocaptate), by coupling the Cs salt of BSH (Cs2-10B12H11SH) with 1-(2-bromoethyl)-2-methyl-5-nitroimidazole followed by purification of the adduct. Imidocaptate was taken up by V-79 cells in culture and showed no inherent toxicity under euoxic conditions up to 1.05 mM (126 micrograms of 10B/mL of culture medium). Imidocaptate showed a dose-dependent decrease in D0 when the treated cells were irradiated with a thermal neutron beam. At the highest dose tested (126 micrograms of 10B/mL of culture medium), the ratio of control to sample D0 values was 2.6 for both linear quadratic and single-hit multitarget models. At 33 micrograms of 10B/mL, imidocaptate showed a control/treated D0 ration (1.5) equal to that observed with the disulfide form of BSH at 28 micrograms of 10B/mL. Compared to BSH and its disulfide, the reduced toxicity and equipotency of imidocaptate suggest that this agent may be useful for boron neutron capture therapy of cancer.

Synthesis, X-ray crystallographic determination, and opioid activity of erythro-5-methylmethadone enantiomers. Evidence which suggests that mu and delta opioid receptors possess different stereochemical requirements.

Enantiomers of erythro-5-methylmethadone (3) were synthesized from optical antipodes of erythro-3-(dimethyl-amino)-2-butanol. X-ray crystallographic analysis of (-)-3 perchlorate revealed that it possesses the 5S,6S absolute configuration. It was found that (-)-3 is substantially more potent than its enantiomer (+)-3 as an opioid agonist in vivo and in vitro. In vitro tests (guinea pig ileal longitudinal muscle and mouse vas deferens preparations) suggest that (-)-3 mediates its effect chiefly through mu opioid receptors. On the other hand, (+)-3 and the more potent enantiomers of methadone, (-)-1, and isomethadone, (-)-2, appear to have less mu-receptor selectivity and interact with a greater fraction of delta receptors than does (-)-3. The fact that the solid-state conformation of (-)-3 differs from that of (-)-1 and (-)-2, which show great similarity in conformational features, suggests that mu and delta receptors have different conformational requirements. The possibility of different modes of interaction with a single opioid receptor population also is discussed.

The binding of 1-(2-hydroxyethyl)-1-nitrosourea to DNA in vitro and to DNA of thymus and marrow in C57BL mice in vivo.

To investigate the hypothesis that the similarity of dose-response curves for induction of thymic lymphoma in C57BL mice was due to similar DNA alkylation profiles for 1-ethyl-1-nitrosourea (ENU) and 1-(2-hydroxyethyl)-1-nitrosourea (HNU), we measured the reaction of the two agents with DNA in vitro and in target tissues in vivo. At equimolar doses, alkylation of DNA by HNU was about 20% greater than that by ENU in vitro. As a percentage of total DNA-bound alkyl groups, relative reaction at a minor groove site (3 of adenine) was similar for the two agents, but HNU caused greater relative alkylation at the major groove sites, O6 and N-7 of guanine. At equi-oncogenic doses, alkylation at the O6 of guanine in liver and thymus was similar for both agents, but O6-alkylguanine formation in bone marrow by HNU was almost twice that by ENU. Because alkylation at O6 of guanine has previously been shown to be a key procarcinogenic lesion in this system, these findings suggest the thymus, rather than the marrow as a primary target for tumor induction by these agents, although involvement of marrow alkylation cannot be ruled out.

Identification of the methyl phosphotriester of thymidylyl (3',5')thymidine as a product from reaction of DNA with the carcinogen N-methyl-N-nitrosourea.

The methyl phosphotriester of thymidylyl(3'-5')thymidine, Tp(Me)T, was obtained as a product of enzymic digestion of N-[14C]methyl-N-nitrosourea-methylated DNA or of N-methyl-N-nitrosourea-methylated [14C]thymine-labelled DNA. The identity of the 14C-labelled Tp(Me)T products was shown by co-chromatography of the 14C-labelled enzymic digests with synthetic Tp(Me)T on Dowex 50 (NH4+ form, eluted at pH 8), and by co-chromatography, on silica gel in 3 solvent systems, of the Tp(Me)T-containing fractions from the Dowex 50 column. This identity was confirmed by showing that the 14C-labelled DNA-derived products hydrolysed in 0.1 M sodium hydroxide at 37 degrees C at a rate identical with that of synthetic Tp(Me)T, and gave the four expected UV-absorbing products (thymidine, thymidylyl(3'-5')thymidine, and the methyl esters of 3'- and 5'-TMP) in the same ratios as the authentic triester.

Evaluation of DNA binding characteristics of some CC-1065 analogs.

The factors influencing the binding of CC-1065 to DNA were examined using racemic analogs with varying chain lengths. The ability of these agents to bind DNA appeared to be related to cytotoxic potency, however this did not appear to be a direct quantitative correlation. Two enantiomers of a bis-indole analog of CC-1065 were studied for DNA binding and cytotoxic activity. The agent with the same stereochemical configuration as CC-1065 was a potent cytotoxin, but its enantiomer was essentially inactive. Both enantiomers showed significant binding to DNA, but the biologically less active isomer showed less overall binding. In all cases, the agents preferred AT-rich DNA, and all bound to similar regions in DNA as evidenced by positions of drug-initiated thermal breaks in single end-labelled fragments of phi X 174RF DNA. The overall similarity in site specificity for binding of the structurally diverse agents suggests that much of the specificity observed in binding of the agent to DNA lies in the DNA itself. Thus, it may be difficult to change minor groove specificity for agents of this type simply by designing structures that can encompass guanine or cytosine residues. Other modifications, such as changing the specificity of the alkylating moiety, may be required to achieve this goal.

The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA).poly(dT).

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.

Cytotoxicity and mutagenicity of aflatoxin dichloride in normal and repair deficient diploid human fibroblasts.

The cytotoxic and mutagenic effect of aflatoxin B1-dichloride (AFB1-Cl2), a direct-acting carcinogen which is a model for the proposed ultimate reactive metabolite of AFB1 (the 2,3-epoxide), was compared in normal, repair-proficient, diploid human fibroblasts and in complementation Group A xeroderma pigmentosum cells (XP12BE) which are virtually incapable of excision repair of DNA damage induced by ultraviolet radiation, the 7,8-diol-9,10-epoxide of benzo[alpha]pyrene, and several reactive aromatic amide derivatives. The XP cells were significantly more sensitive than normal to the cytotoxic and mutagenic effects of AFB1-Cl2, not only as a function of concentration administered but also of the number of AFB1-Cl2 residues initially bound to DNA. Cytotoxicity was determined from survival of colony-forming ability; resistance to 6-thioguanine was the genetic marker used for mutagenicity. We compared the rate of loss of AFB1-Cl2-DNA adducts from cells treated and held in the non-dividing state (confluent) over several days, as well as their ability to recover from the potentially mutagenic and/or cytotoxic effects of the agent. AFB1-Cl2 residues were lost from both strains of cells and both exhibited a gradual increase in survival. However, the rate of loss of adducts from the DNA in the normal cells was more rapid than in XP cells and they exhibited recovery from higher doses of AFB1-Cl2 than XP cells. The major primary DNA adduct formed in the human cells and in isolated DNA was a chemically unstable guanine derivative which could undergo a change in structure with time posttreatment to form a more stable secondary adduct. The cytotoxic effect of AFB1-Cl2 was highly correlated with the presence of either of these guanine adducts. Evidence suggests that the primary adduct is an N7-guanine adduct. The kinetics of the loss of this guanine and its transformation into the more stable secondary adduct resembled that reported recently for the major primary DNA adduct formed by the reaction of AFB1 at the N-7 position of guanine in the DNA of normal and XP cells and its transformation into the putative AFB1-ring opened triamino pyrimidyl structure.

The Salmonella mutagenicity test: evaluation of 29 drug candidates.

The Salmonella mutagenicity test (Ames assay) is part of the routine screening battery applied to all new drugs at The Upjohn Company. The purpose of this paper is to report results for 29 compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of known biological activity and other reasons. None of the compounds reported here produced an increase in revertant colonies in the Salmonella strains employed (TA98, TA100, TA1535, TA1537 and TA1538) and therefore the Salmonella mutagenicity results with these materials do not suggest potential for mutagenesis or carcinogenesis.

Genotoxicity of tacrine in primary hepatocytes isolated from B6C3F1 mice and aged ad libitum and calorie restricted Fischer 344 rats.

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine; THA), a reversible centrally acting anticholinesterase, has been shown to be potentially useful for treatment of patients with Alzheimer's disease. However, currently available forms of THA may be therapeutically limited by the fact that high doses have resulted in liver and kidney damage. To determine if THA is hepatotoxic via a genotoxic mechanism, we evaluated its ability to induce unscheduled DNA synthesis (UDS) in primary cultures of rodent hepatocytes. Positive dose-dependent increases in UDS were observed in hepatocytes derived from male B6C3F1 mice and from young, middle-aged, old, and old Aroclor-induced (ARO) male F344 rats maintained on either an ad libitum (AL) or a caloric restricted (CR) diet (60% of AL) and exposed to 0.05-1000.0 micrograms/ml of THA. Hepatocytes from old AL rats, treated with THA, exhibited significant age-related decreases in DNA repair compared to young and middle-aged AL rats. By contrast, cultures from CR rats exhibited age- and diet-related decreases in UDS from the AL and young CR animals, respectively. Moreover, ARO-induced old AL- and CR-derived hepatocytes exhibited significant increases in UDS compared to uninduced old AL and CR animals. No cytotoxicity was observed in the uninduced old AL- or any CR-derived hepatocytes. These data indicate that the aged and CR fed animal is less susceptible to the cytotoxic and genotoxic effects of THA; while the younger AL fed and enzyme induced old AL or CR fed animals were more susceptible. The data suggest that THA may be a genotoxic rodent carcinogen. At present, the relationship of these findings to the clinical use of THA are unclear and further study is required.

Clinical assessment of trunk flexor muscle strength in healthy girls 3 to 7 years of age.

We developed a clinical method for assessing trunk flexor muscle strength in healthy children that can be applied to assist physical therapists in determining strength accurately in pediatric patients. In this study, we assessed trunk flexor muscle strength in 75 healthy girls 3 to 7 years of age. Muscle strength was graded on a scale of 0 to 5 using modified, manual muscle testing methods. These methods attempted to minimize the amount of hip flexor muscle activity during trunk flexion and allow more isolated action of the abdominal trunk flexor muscles. The frequency of Normal (Grade 5) strength first appeared to predominate at age 5 years with the majority of children demonstrating Normal (Grade 5) strength by age 7 years. Regression analysis illustrated a positive linear relationship (beta = .37, p less than .001) between mean muscle grade and age group. We discuss muscle cross-sectional area, muscle-fiber diameter, muscle-cell number, maturation of the central nervous system, and changes in body proportions with age as possible contributing factors to the results of this study.