Molecular analysis of 5-azacytidine-induced variants in mammalian cells.

5-azacytidine induces 6-thioguanine resistance in AS52 cells. To characterize these resistant clones, we isolated 148 of them from 50 independently treated flasks. Less than nine (6%) of the 148 variants were spontaneous. PCR amplification of the DNA primers flanking the gpt gene produced no product in 15 clones (10%). Of the 133 remaining clones, 52 showed sequence alterations in the gpt structural gene. Of these 52, 34 (65%) were GC-->CG transversions. Only seven were located in CpG sequences. Thus, methyltransferase complexes are not major contributors to 5-azacytidine-induced point mutations in AS52 cells. The remaining 81 clones had no sequence alterations within the coding region of the gpt gene. Southern blot analysis of a sample of these variants (37/81) indicated that the 6-thioguanine-resistant phenotype was not due to local rearrangements or deletions (resolution 50 bp). Sequence analysis of the early promoter region of another sample of these variants (24/81) indicated that lesions in the promoter could not be responsible for the 6-thioguanine resistance observed. Thus, a majority of these variants were formed via a mechanism other than small genomic rearrangements, point mutations or deletions of the gpt structural gene or its promoter. Neither the mechanisms leading to these variants nor the biological and morphological consequences of these variants are known.

The anatomical basis of sciatica secondary to herniated lumbar disc: a review.

The purpose of this manuscript is to illustrate the key anatomical and biomechanical elements involved in the etiopathogenesis of sciatica, and to demonstrate how periradicular fibrosis contributes to the pathophysiology of recurrent post-operative sciatica. History, etiology, anatomy and diagnosis of herniated inter-vertebral disc are reviewed. The straight leg raising exam is a well accepted test in the diagnosis of lumbar disc herniation. In the post-operative patients, the results of the straight leg raising test are affected by the presence of scar and fibrosis around the lumbar root(s) involved. The mechanisms by which perineural fibrosis and adhesions change and compromise the neural dynamics and causes symptoms to recur in the post-operative patient are discussed. Due to its dramatic clinical relevance, prevention of periradicular fibrosis has high priority in the surgical management of herniated lumbar disc. Such a goal should be obtained by using a combination of appropriate indication to surgery, impeccable operative technique and the use of an effective anti-fibrotic agent.

Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) results in a broad spectrum of biological responses, including altered metabolism, disruption of normal hormone signaling pathways, reproductive and developmental effects, and cancer. Cytochrome P450 1B1 (CYP1B1) is a dioxin-inducible gene that is active in the formation of 4-hydroxyestradiol, a potentially genotoxic catechol estrogen. Therefore, the analysis of CYP1B1 in humans may be useful in establishing relationships between dioxin exposure and adverse health effects. In this study, we examined the expression of CYP1B1 in human peripheral blood lymphocytes of unexposed individuals using a quantitative reverse transcription-PCR method. Absolute CYP1B1 RNA levels varied more than 30-fold in uncultured mononuclear cells obtained from 10 individuals. In vitro treatment of mitogen-stimulated lymphocytes with TCDD for 1-5 days of culture resulted in a peak induction of CYP1B1 after 3 days. The induction of CYP1B1 RNA levels after 3 days of culture was dose-dependent, exhibited a maximum response above 10 nM TCDD, and varied greatly among different individuals. However, the half maximal dose required for this induction was similar between individuals and comparable to that observed in the MCF-7 and HepG2 human cell lines. These observations indicate that CYP1B1 exhibits variable constitutive expression and is inducible in vitro by TCDD in human lymphocytes and that the magnitude of induction varies within the population. These data define the suitability of CYP1B1 for use as a mechanistically based biomarker in ongoing molecular epidemiological studies of human populations exposed to dioxins and related chemicals that bind the aromatic hydrocarbon receptor.

Analysis of large and small colony L5178Y tk-/- mouse lymphoma mutants by loss of heterozygosity (LOH) and by whole chromosome 11 painting: detection of recombination.

Analysis of 122 spontaneous large and small colony mutants derived from L5178Y tk +/- mouse lymphoma cells at 28 heteromorphic microsatellite loci on chromosome 11 showed that extensive loss of heterozygosity (LOH) is common in both large colony and small colony mutants, eliminating most chromosome 11 loci as candidates for a putative growth control locus. These results, in conjunction with historical cytogenetic data, suggest that a putative growth control locus lies distal to the thymidine kinase (Tk1) gene, near the telomere. Thirty seven mutants were hybridized with a chromosome 11-specific whole chromosome painting probe for analysis of rearrangements. Generally, painting confirmed earlier observations that large colony mutants are karyotypically normal, whereas small colony mutants frequently have detectable rearrangements. A point probe distal to Tk1 revealed no evidence of chromosome breakage in small colony mutants that appeared normal on whole 11 painting and had no LOH. Therefore, the molecular difference between large and small colony mutants remains unknown. Models to explain large and small colony mutants consistent with our findings are presented, including loss of a putative growth control gene, differential mechanisms of chromosome breakage/repair and second site mutations as explanations for small colony mutants. Painting revealed translocations and aneuploidy and showed that non-disjunction was not a common explanation for complete LOH. The most common finding was that large regions of LOH do not result from deletions, demonstrating that these cells can detect recombination events as well as previously observed chromosomal rearrangements, deletions and point mutations.

Active noise reduction versus conventional hearing protection. Relative benefits for normal-hearing and impaired listeners.

The benefits of active noise reduction (ANR) hearing protectors were assessed in two groups of normal-hearing subjects, under and over the age of 40 years, and one group with bilateral high-tone hearing loss. Subjects were tested with the ears unoccluded and fitted with conventional sound attenuating E-A-R foam plugs, E-A-R HI-FI plugs, and Bilsom Viking muffs; and one ANR muff, the Peltor 7004. Within each ear condition, measurements were made in quiet of hearing thresholds for frequencies between 0.25 kHz and 8 kHz, duration and frequency difference limens, and word recognition. Hearing thresholds and word recognition were also measured in a background of impulsive cable swager noise. The E-A-R foam plug provided the highest and the E-A-R HI-FI plug, the lowest attenuation. The Bilsom Viking and Peltor muffs were virtually identical and midway between. An additional 10 dB of sound reduction was realized at 0.25 kHz with ANR. The masking effect of the noise on hearing threshold decreased with an increase in attenuation. None of the devices compromised either duration or frequency discrimination. Word recognition in noise improved in normal listeners when protectors were worn. For the impaired subjects, word recognition with poor contextual cues decreased with an increase in sound attenuation, in both quiet and noise. Like older normal listeners, their scores were relatively higher with ANR.

Design of a survey to inform state health decision making: a collaborative effort.

BACKGROUND: The North Carolina Health Profile (NCHP), a statewide telephone survey, was introduced as part of the state's Health Policy Information Project aimed at enhancing the use of health data for state policy decision making and program management. A key factor in the creation of the NCHP was a collaboration between the State Center for Health Statistics and the Survey Research Unit at the University of North Carolina at Chapel Hill. The purpose of this article is to describe our partnership, the development of the survey design, and the dissemination of survey results. METHODS: Three designs were considered during the planning and development of the survey. The final design consisted of a random digit dialing sample of 2,400 households in the state's noninstitutionalized population. The questionnaire was comprised of an adult module (addressing adult health care use and insurance coverage), a child module (addressing health care use and insurance coverage of children ages 0-17 years), and a young child module (addressing child development and safety for children ages 0-5 years). RESULTS: Several statistical briefs, a report, a public dataset, and accompanying public use documentation were prepared for a variety of audiences, including state legislative committees and commissions, state agencies, and advocacy groups. DISCUSSION: We learned several lessons in our research and practice partnership including the need for collaboration between data creators and users, for addressing obstacles in soliciting policy information needs, and for prioritization in meeting information needs.

5-Azacytidine-induced 6-thioguanine resistance at the gpt locus in AS52 cells: cellular response.

Treatment of AS52 cells with 5-azacytidine resulted in an induction of 6-thioguanine-resistant [6TG] colonies, which reached a maximum by an expression time of 9 days. Dose responses for both cytotoxicity and mutation induction were determined following treatment with 5-azacytidine. At 20 microM treatment, 5-azacytidine exposure resulted in about 50% survival. Mutant frequency reached a maximum of 10 microM. At concentrations between 10 and 20 microM, 5-azacytidine was a potent mutagen but did not exhibit a dose response. Although many compounds both induce cell death and affect the growth rate of cells, 5-azacytidine specifically induced cell death and did not affect the doubling time of the surviving treated cell population.

Micronuclei induced by modulators of methylation: analogs of 5-azacytidine.

Jones and coworkers demonstrated a qualitative correlation between 5-azacytidine and some of its analogs in inducing changes in cell morphology and their ability in preventing DNA methylation. Previously, we evaluated the same compounds to determine their ability to induce trifluorothymidine (TFT) resistance in L5178Y mouse cells and found that their mutagenic potency also correlated with their reported ability to induce morphological changes in C3H10T1/2 cells. Here, we examine four of the same analogs, 5-fluoro-2'-deoxycytidine, 5-azacytidine, 5,6-dihydro-5-azacytidine and 6-azacytidine, to find out if micronuclei induced by these compounds correlated with these effects. The most cytotoxic analog was 5-fluoro-2'-deoxycytidine, followed by 5-azacytidine. 5,6-Dihydro-5-azacytidine and 6-azacytidine were substantially less cytotoxic. All four compounds induced micronuclei. The lowest dose ranges at which responses were observed for micronucleus induction were -0.04 microM for 5-fluoro-2'-deoxycytidine, 0.2 microM for 5-azacytidine and 10-20 microM for 5,6-dihydro-5-azacytidine and 6-azacytidine. Lack of kinetochore staining in most of the micronuclei indicated that all four compounds were clastogenic. We note a general trend in the biological activity of these analogs: compounds that are specifically blocked at the 5 position such as 5-azacytidine and 5-fluoro-2'-deoxycytidine effect changes in cell morphology, cytotoxicity, TFT resistance and the induction of micronuclei at very low doses. 5-Azacytidine analogs that possess more chemically accessible 5 positions such as 5,6-dihydro-5-azacytidine and 6-azacytidine either require doses that are orders of magnitude greater to induce these effects or are unable to induce changes in cell morphology and TFT resistance at doses below which the compound is lethal to the cells.

In situ and suspension protocols for chemically-induced mutation at the tk locus in L5178Y MOLY cells: dose response and colony size distribution.

We used EMS up to concentrations of 0.25 microliters/ml (292 micrograms/ml) to induce mutations at the tk locus in L5178Y MOLY cells, measured the cellular response by the in situ mutation assay protocol and compared these results to those obtained in a concomitant suspension assay. EMS induced mutagenic responses with both protocols. The mutant fraction for the solvent control was 89 mutants per million viable colonies for the suspension protocol and 426 mutations per million viable cells plated for the in situ protocol. These numbers increase to 447 and 2073 respectively, with 0.25 microliter/ml EMS treatment. Sizing curves indicated that the in situ protocol detected a greater proportion of smaller colonies than did the suspension protocol. Not only were the number of small colonies greater than large colonies in the in situ protocol, but their rate of increase was also slightly higher than that of the large colonies. The in situ protocol also reduces the time and cost of experimentally performing the assay compared to the suspension protocol. In this paper we compare the use of the suspension and in situ protocols to measure chemically-induced mutations and demonstrate that the latter method detects a larger fraction of induced mutations at the tk locus in L5178Y MOLY cells.

Is micronucleus induction by aneugens an early event leading to mutagenesis?

This study was designed to investigate a previously unidentified potential mechanism for mutation induction as well as to clarify a biological consequence of micronuclei with mutation induction as measured by trifluorothymidine (TFT) resistance in mouse L5178Y cells using four aneugens: colcemid, diethylstilbestrol, griseofulvin and vinblastine. All four compounds induced micronuclei which appeared in the first cell cycle after treatment. More than 85% of the micronuclei induced by each compound stained positive for the presence of kinetochores implying that the micronuclei contained whole chromosomes. However, these same compounds were unable to induce TFT resistance under three different treatment regimes. We concluded that these compounds, under conditions where they induce primarily kinetochore positive micronuclei, were not able to induce mutations. Thus, the induction of micronuclei containing whole chromosomes harboring a selectable gene is not an early event leading to mutations in these cells.

An in situ protocol for measuring the expression of chemically-induced mutations in mammalian cells.

The generation of expression curves and the evaluation of mutagenic responses of mammalian cells using standard mutagenesis assays can be inaccurate because mutant and wild-type cells are usually mixed during the expression phase. If some mutant progenitors or mutants grow more slowly than the wild-type cells during the expression period, there will be a decrease in the mutant to wild-type ratio with time and the mutant fraction will not accurately represent the number of mutational events that occurred. The mutant fraction may also inaccurately assess the number of mutations if these mutations are expressed over a number of generations during the time before selection. We previously showed that recovery of L5178Y mouse cell mutants is not complete when mutations are allowed to express in suspension because slowly growing mutants and/or mutant progenitors are diluted out during this time (Rudd et al., 1990). In order to more accurately quantitate the mutagenic response of the cells, we developed an in situ procedure which segregates and immobilizes cells during expression. Because of this immobilization, slowly growing mutant progenitors and mutants expressed at different times will have an equal probability of being scored as mutants. Thus, one mutation leads to one mutant colony and the measurement of the mutagenic response of the cells to the chemical accurately reflects the mutational events that occurred. We plated L5178Y tk+/- mouse cells in semisolid medium immediately after treatment. As the cells grew and formed microcolonies, the selective agent TFT was added as an overlay at specified times, permitting only TFTr cells to survive. In this procedure, each mutation was captured as an individual colony; consequently, the measured mutation fraction accurately reflected the mutational events that occurred at the selected locus. In addition, the induced mutant colonies arising in the agar are the result of independent mutational events. We previously described the in situ protocol for L5178Y cells and showed that the spontaneous mutation rate measured was 50-fold greater than when the cells expressed the phenotype in suspension (Rudd et al., 1990). From this we concluded that the slow growth phenotype was expressed before TFT resistance. In the present paper, we evaluate the effect of chemical treatment on the mutation fraction as a function of the time to TFT addition. Using the in situ protocol, we generated expression curves for three nucleotide analogs, 5-azacytidine, TFT and AraC. The numbers of TFTr colonies produced at various times after treatment indicated that chemically-treated cultures had higher mutation fractions than the solvent controls.(ABSTRACT TRUNCATED AT 400 WORDS)

An investigation of micronucleus and mutation induction by oxazepam in mammalian cells.

The benzodiazepines are a class of drugs that are widely used in the treatment of various psychiatric disorders. One member of this class, oxazepam, is also a common metabolite of several other benzodiazepines. Since the evidence for the genetic toxicity and carcinogenic properties of these compounds is inconsistent, we investigated the oxazepam-induced formation of micronuclei in Syrian Hamster embryo fibroblast (SHE) cells, human amniotic fluid fibroblast-like (AFFL) cells and L5178Y mouse cells. A dose-dependent increase in micronucleus fractions was found in all three cell lines. The time course of micronucleus induction in L5178Y cells showed a maximum at 5 h after treatment, suggesting that the micronuclei were formed in the first mitosis after treatment. Kinetochore staining (CREST-antiserum) revealed the presence of kinetochores in approximately 50% of the micronuclei in all three cell types. This result was further confirmed by in situ hybridization in L5178Y cells and indicates the presence of whole chromosomes or centric fragments as well as acentric fragments in the oxazepam-induced micronuclei. The L5178Y cells did not show a mutagenic response to oxazepam at any of the doses or expression times used.

The use of sublaminar cables to replace Luque wires.

Sublaminar wires have been used in conjunction with posterior instrumentation to stabilize the spine. Sublaminar wiring has fallen into disfavor because of an increase in neurologic complications with the Luque technique as well as wire breakage, dural tears, and difficulty of removal. A cable system consisting of two 49-stranded stainless steel cables connected to one malleable leader was designed to overcome these shortcomings. Biomechanical testing revealed that the maximum yield strength of a single stainless steel cable loop was 2.85-2.94 times greater than a double 0.05-in. stainless steel wire loop. The fatigue tests demonstrated that the stainless steel cables required 6-22 times more cycles to failure than the stainless steel wire. Many of the titanium cables failed immediately under higher loads (0-100 lb) because of slipping of the crimp. The preliminary clinical results after a mean of 19 months of follow-up of 245 cables are encouraging. There has been no breakage or loosening of the cables and no complications associated with the use of the cables. The stainless steel cables are very strong, but more important, the cable flexibility prevents repeated contusions to the spinal cord during insertion of the rods and tightening of wires. The cable conforms to the undersurface of the lamina. This may lead to a decrease in neurologic complications.

Effects of sodium hyaluronate on peridural fibrosis after lumbar laminotomy and discectomy.

Sodium hyaluronate, 1.9% solution, was evaluated for its ability to retard peridural fibrosis after unilateral lumbar hemilaminotomy, anular fenestration, and nuclectomy in dogs. Three materials: fat grafts, gelfoam, and sodium hyaluronate, were compared with empty controls for their ability to inhibit peridural fibrosis. Each dog served as his own internal control and the formation of fibrosis was evaluated at 2, 4, 12, and 26 weeks. Sodium hyaluronate was found to inhibit fibrosis more than the other materials on both a macroscopic and microscopic level. The area of fibrosis and tenacity of the adhesions on dissection were notably less in the sodium hyaluronate group. Microscopically, the thickness of collagen and number of fibroblasts were decreased with the use of 1.9% sodium hyaluronate. The peridural fibrosis occurred equally both anteriorly and posteriorly to the nerve roots and correlated with the area of surgical dissection. Fat grafts were not effective in preventing fibrosis anteriorly, especially in the region of the exiting nerve roots. Gelfoam did not inhibit but actually appeared to increase fibrosis formation. Interposition materials currently used in humans to prevent scar formation such as gelfoam and fat grafts have only addressed the posterior scar formation, which do little to alter the fibrosis anteriorly. The adhesions between the nerve root and the anulus fibrosus bind the nerve root down anteriorly, making it more vulnerable to recurrent disc herniation. Sodium hyaluronate, 1.9% solution, with its viscous semifluid properties, coats the nerve roots and dura anteriorly and posteriorly.(ABSTRACT TRUNCATED AT 250 WORDS)

The practicing doctor's perspective on the surgical curriculum.

The purpose of this study was to identify deficiencies of surgical clerkship and residency curricula in the training of family physicians. Responses to a survey from 202 practicing family practitioners were analyzed. Orthopedics, otolaryngology, urology, neurosurgery, and cardiovascular surgery were surgical specialty areas where more than 40 percent of the respondents thought they had spent too little time. Learning objectives were either not presented or were used ineffectively, according to the majority of respondents. Fifty percent believed that indicating a career interest in family medicine resulted in a negative bias toward them during the surgery clerkship. Over 45 percent thought they were moderately or totally unprepared at the completion of their training to perform several basic surgical procedures. The results of this survey indicate the need for an increased allocation of curricular time to several surgical specialties and an augmented emphasis on outpatient experiences within the surgical curriculum.

The effect of an inpatient chemical dependency rotation on residents' clinical behavior.

The effect of a rotation in a chemical dependency unit on subsequent resident diagnoses in the family practice clinic was investigated by clinic audit. A significant increase in diagnoses of alcoholism (56 vs. 18, P less than .05) and chemical dependency (114 vs. 49, P less than .001) was found in the year following initiation of the rotation. An inpatient rotation immediately increases resident recognition of addictive illness in the model clinic.

Changes in supply and distribution of family physicians in the United States.

From 1970 to 1980, the supply of family and general physicians in the United States increased by 4 percent. The overall increase was not felt uniformly among the states. Rather, the distribution reflected general regional trends in the United States. The analysis derives from a comparison of 1970 and 1980 American Medical Association and Bureau of the Census data. A study is made of changes in the supply of family and general physicians, in the number of residents in family practice programs, in the supply of general internists and pediatricians, in the population, and in the per capita income of each state. Regions with economic and population growth also benefited from immigration of family physicians and from new residency programs. They had fewer barriers to growth in the form of primary care competitors and elderly general practitioners requiring replacement. The dominance of market forces in channeling the effects of educational and manpower politics raises challenges for the specialty of family practice.

Alcoholic facial neuralgia: report of three cases.

Three cases of jaw pain recurrently precipitated by consumption of alcohol are described. Alcohol-induced neuralgia is added to the differential diagnosis of atypical orofacial pain.

The supply of physicians in four midwestern states.

At a time when national policies find their basis in a projected physician surplus, the specialty of family medicine must continue to strive for growth. Organizations such as the American Academy of Family Physicians (AAFP) provide support and defense for issues essential to the growth and survival of family medicine. The strength of the Academy resides in an active and growing membership, and concerns about membership should be identified and addressed. This paper describes the AAFP membership in four midwestern states: Illinois, Iowa, Indiana, and Wisconsin. The age distribution and medical school location patterns of members are presented. In Illinois, almost half the members either currently are or will be 65 years or older within the decade. In the other three states, the comparable pool is only 38%. In Illinois, half the members attended an Illinois medical school and one-third are foreign medical school graduates. In Indiana and Iowa, the vast majority of members are graduates of Indiana and Iowa medical schools, respectively. Wisconsin profits from both its own graduates and from the attraction of physicians trained in other states. Discussion focuses on the needs facing the chapters and on variables that might be important for increasing membership.