Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro.

Previously, Alternaria extract and metabolite mutagenicities+/-nitrosylation were characterized using Ames Salmonella strains TA98 and TA100, which are both reverted at GC sites. To examine other targets for mutation, the metabolites Altertoxin I (ATX I), Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME), Tentoxin (TENT), Tenuazonic acid (TA) and Radicinin (RAD) were reexamined+/-nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run of C's, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more sensitive to oxidative damage. ATX I was also assessed for mammalian mutagenicity at the Hprt gene locus in Chinese hamster V79 lung fibroblasts and rat hepatoma H4IIE cells. When tested from 1 to 100 microg/plate without nitrosylation, ATX I was mutagenic in TA102+/-rat liver S9 for activation and weakly mutagenic in TA104+/-S9, demonstrating direct-acting AT base pair mutagenicity. AOH was also directly mutagenic at AT sites in TA102+/-S9 while AME was weakly mutagenic in TA102+/-S9 and TA104+S9. Nitrosylation of ATX I enhanced mutagenicity at AT sites in TA104+/-S9 but produced little change in TA102+/-S9 compared to native ATX I. However, nitrosylated ATX I generated a potent direct-acting frameshift mutagen at C sites in TA97+/-S9. While ATX I was not mutagenic in either V79 cells or H4IIE cells, 5 and 10 microg/ml nitrosylated ATX I produced a doubling of 6-thioguanine resistant V79 colonies and 0.5 and 1 microg/ml were mutagenic to H4IIE cells, becoming toxic at higher concentrations. These results suggest ATX I, AME and AOH induce mutations at AT sites, possibly through oxidative damage, with nitrosylation enhancing ATX I frameshift mutagenicity at runs of C's. Nitrosylated ATX I was also directly mutagenic in mammalian test systems.

Adaptation of the WHO guideline for residual DNA in parenteral vaccines produced on continuous cell lines to a limit for oral vaccines.

Although there is a WHO guidance for a limit on residual DNA for parenterally administered vaccines produced on continuous cell lines, there is no corresponding guidance for oral vaccines. To help determine an oral limit, we performed a study of Vero cell DNA uptake in rats, in which the relative uptake and persistence of Vero cell DNA administered orally was compared to its uptake when delivered intramuscularly (IM). The results of this study allowed the generation of an empirically derived IM versus oral factor (10(6)) representing the relative inefficiency of DNA uptake by oral administration. This factor was then applied to the WHO recommended parenteral limit of 10 ng/dose to determine a corresponding upper limit on the level of residual Vero cell DNA for an oral vaccine of 10 mg. As a conservative approach, this empirically determined limit was reduced 100-fold to 100 microg. Thus, the results of this animal study, together with additional evidence in the literature, support a residual DNA safety limit of 100 microg per dose for an oral vaccine produced on a continuous cell line.

International variations in epidemiology of cancers of the extrahepatic biliary tract.

Previous studies of the descriptive epidemiology of biliary tract cancers have not differentiated among different types of biliary tract cancer because until recently the International Classification of Diseases (ICD) did not classify them separately. Recent versions of the ICD now distinguish cancers of the gallbladder, extrahepatic bile ducts, and ampulla of Vater. In order to describe more precisely the distribution of these three cancers, we obtained data from nine cancer registries throughout the world which used the eighth or ninth revision of the ICD. Sex-specific, age-adjusted disease rates were calculated for each disease. Log-linear models were used to evaluate the association of age and sex with the risk of acquiring each disease and to assess whether the risk of acquiring disease or the age and sex distribution of the three diseases varied by geographic location. Gallbladder cancer was the most common of the three diseases and occurred more frequently in females. Extrahepatic bile duct cancer was the next most common disease and occurred equally in both sexes. Cancer of the ampulla of Vater was the least common and was more common in males. The incidence of each of the diseases increased with age. The age and sex distributions of the different diseases different among the nine registries. Thus these three neoplasms differ in their descriptive epidemiology and should therefore be considered separately in clinical practice and in future investigations.

The association of nonsteroidal anti-inflammatory drugs with upper gastrointestinal tract bleeding.

To evaluate the risk of developing upper gastrointestinal (UGI) bleeding from nonsteroidal anti-inflammatory drugs (NSAIDs), a retrospective (historical) cohort study was performed, using a computerized data base including 1980 billing data from all Medicaid patients in the states of Michigan and Minnesota. Comparing 47,136 exposed patients to 44,634 unexposed patients, the unadjusted relative risk for developing UGI bleeding 30 days after exposure to a NSAID was 1.5 (95% confidence interval 1.2 to 2.0). Univariate analyses demonstrated associations between UGI bleeding and age, sex, state, alcohol-related diagnoses, preexisting abdominal conditions, and use of anticoagulants. This association between NSAIDs and UGI bleeding was unchanged after adjusting for these potential confounding variables using logistic regression. A linear dose-response relationship and a quadratic duration-response relationship were demonstrated. Non-steroidal anti-inflammatory drugs are associated with UGI bleeding, although the magnitude of the increased risk is reassuringly small.

The relative gastrointestinal toxicity of the nonsteroidal anti-inflammatory drugs.

To assess the relative rate or upper gastrointestinal (UGI) tract bleeding associated with nonsteroidal anti-inflammatory drugs (NSAIDs), we performed a retrospective cohort study using 1980 billing data from all Medicaid patients in the states of Michigan and Minnesota. The rate of UGI tract bleeding in the 30 days following each drug exposure was examined in the 88,044 patients dispensed only one of seven NSAIDs. The rate of UGI tract bleeding differed significantly among users of these drugs. Stratification and logistic regression were used to adjust for multiple potential confounding factors, without substantive changes in the results. An alcohol-drug interaction was found. Sulindac users had the highest rate of UGI tract bleeding, and it was the only drug statistically different from ibuprofen. When the average daily dose of sulindac received was divided by the maximum recommended daily dose, it was notably higher than those for other drugs. Repeated analyses using data from 1982 confirmed these results. We conclude that there are significant and consistent differences in the incidence of UGI tract bleeding associated with the use of NSAIDs in this population.

Upper gastrointestinal tract bleeding from oral potassium chloride. Comparative risk from microencapsulated vs wax-matrix formulations.

A retrospective cohort study was performed to assess the relative risk of upper gastrointestinal (UGI) tract bleeding from two formulations of potassium chloride. Relevant information was obtained from 1980 through 1984 Medicaid billing data from the states of Michigan, Minnesota, Florida, and Ohio. After patients with a history of UGI tract bleeding prior to their first prescription for either of the two potassium chloride preparations under study were excluded, data were analyzed for 28,790 patients (143,512 patient-months) dispensed a microencapsulated formulation exclusively and 76,118 patients (560,341 patient-months) dispensed a wax-matrix formulation exclusively. The risk of UGI tract bleeding within 30 days after each prescription for the drug of interest was examined. After sampling from the undiseased study subjects and adjusting for multiple potential confounding variables using logistic regression, an odds ratio (95% confidence interval) of 0.67 (0.52 to 0.85) was observed.

Investigation of the source of the blue field entoptic phenomenon.

The cellular source of the blue field entoptic phenomenon was investigated in two microvascular preparations using video-microscopy with lighting conditions similar to those under which the entoptic phenomenon is visualized within the human eye. In the wing of the hibernating bat, microvascular flow was simultaneously videotaped under transmission illumination at 430 nm and under unfiltered illumination. In the rat cremaster alternating observations were made using transmission illumination at 430 nm and epi-illumination fluorescence microscopy with leukocytes rendered fluorescent by intravenous Quinacrine. In both preparations, low magnification video-microscopy using 430 nm illumination produced a field of particles, which were brighter than the background, flowing within a network of dark vessels. The appearance of the particles and their movement simulated the blue field entoptic particle motion. Under higher magnification, the particles appeared brighter than the plasma gaps between red blood cells and were demonstrated to be leukocytes by morphology, by specific staining and by typical behavioral movement. The particles were observed in terminal arteriols capillaries, and post-capillary venules where they were not obscured by red blood cells. The results of this study of two microvascular preparations strongly suggest that in the human eye the blue field entoptic phenomenon is produced by leukocytes flowing within the macular retinal microvasculature.

Cancer incidence in a group of workers potentially exposed to ethylene oxide.

BACKGROUND: Cytogenetic changes associated with ethylene oxide (ETO) exposure at a worksite prompted a study of cancer incidence in that cohort. METHOD: Cancer incidence through 31 December 1987 was ascertained in a cohort of 1132 individuals employed at the worksite at any time from 1 July 1974 through 30 September 1980, the period of potential exposure to ETO at the plant. The number of observed cancers was compared with that expected based on age- and sex-specific incidence rates reported by the National Cancer Institute's Surveillance Epidemiology and End Results Program. Standardized morbidity ratios (SMR) were calculated separately for regular and temporary employees. RESULTS: Of the 28 cancers observed in the cohort, 12 were breast cancers. The SMR for breast cancer among regular female employees ranged from 2.55 (95% CI: 1.31-4.98, P = 0.02) to 1.70 (95% CI: 0.89-3.23, P = 0.09) depending on calendar year of follow-up, assumptions about completeness of follow-up, and the reference rates used. The excess of breast cancer over expected in regular female employees diminished over time. No statistically significant excess of breast cancer was noted for temporary female employees at any point during follow-up. No increase in cancer incidence was found over that expected for any cancer sites associated with ETO in previous studies--leukaemia, brain, pancreas and stomach. CONCLUSIONS: Factors such as appropriateness of latency periods, length of follow-up and lack of a common histopathological type need to be considered in evaluating the excesses in observed breast cancer incidence, which diminished over time.

Health and medical care for the elderly and aged population: the state of the evidence.

Emerging statistics emphasize the special needs of the elderly for health and medical care services. A variety of services are being recommended to meet these needs, but few of them have been subjected to rigorous study for determining efficacy, effectiveness or cost. Examination of about 1,000 studies of health and medical services for the elderly published between 1967 and 1978 revealed only 7 that met predetermined criteria for well-designed and executed investigations. The varied needs of the elderly are finally being recognized, and undoubtedly pressure will be increased for meeting these needs. In this era of finite monetary resources and budgetary restraints, we need better data upon which to base the selection of medical care services for the aged. Resources needed to provide care of known benefit should not be diverted to provide care of unknown benefit.

The heart rate-corrected QT interval of conscious beagle dogs: a formula based on analysis of covariance.

Three frequently used and cited formulas used to rate correct the QT interval (Bazett's, Fridericia's, and Van de Water's) were compared and ranked using a large population-based cohort of beagle dogs (99 males and 99 females). In addition, analysis of covariance was used to derive a flexible method to rate correct the QT.interval for heart rate. The method is flexible in that it utilizes pretest or control data to determine the degree of correction. In addition, it can also be used to evaluate whether treatment alters the association between heart rate and QT. Specifically, pretest QT (unadjusted) and heart rate data were used to estimate coefficients in the linear regression log(QT) = alpha + beta log(HR). The estimated slope (beta) from the pretest data was used to heart rate correct the QT interval in the formula log(QT)ca = log(QT) - beta *[log(HR - log(HRm)]. The term "log(HRm)" is included to standardize QTca to a reference value, either a fixed value or an average heart rate for the data set being analyzed. These formulas were retrospectively compared under a typical toxicity study paradigm with a class III antiarrhythmic agent (L-768,673) that selectively prolongs the QT interval by blocking the slow activating component of the delayed rectifying potassium channel (lks). Based on their ability to dissociate the effects of heart rate on the QT interval, the formulas received the following ranking: Covariate Adjustment (preferred) = Van De Water's > Fridericia's > Bazett's (not recommended). Analysis of covariance based on pretest or control data is preferred for moderate to large studies where there are adequate data for estimation of the slope parameter beta, the investigator does not have sufficient control over HR, or treatment alters the association between HR and the QT interval. Conversely, for smaller studies a fixed rate adjustment formula from the literature (such as Van de Water's or Fridericia's equations) may be preferable since the bias from using a fixed formula is likely to be smaller than the variance resulting from estimating beta from a small sample.

Diet, caloric restriction, and the rodent bioassay.

The diet can significantly alter the results of toxicity and carcinogenicity studies. Ad libitum (AL) overfeeding of excessive calories to sedentary adult rodents is one of the most poorly controlled variables affecting the current rodent bioassay. AL-overfed rodents develop an early onset of adverse metabolic events, endocrine-disruptive degenerative diseases, and tumors that result in early morbidity and mortality. AL food consumption is extremely variable, but has a strong correlation with adult body weight, obesity, and survival. AL feeding of diets with modified protein, fiber, and energy content are not as effective as simple, moderate dietary (caloric) restriction (DR) in controlling these study variables. Moderate DR (70-75% of adult AL) is operationally simple and controls adult body weights, prevents obesity, and improves health and survival by reducing or delaying diet-related endocrine, renal, and cardiac diseases. Moderate DR provides a uniform rodent model, increases treatment exposure time, and increases the statistical sensitivity of these chronic bioassays to detect true treatment effects. Feeding a balanced diet by a moderate DR regimen of 70-75% of the maximum, unrestricted adult AL food intake is recommended for conducting well-controlled toxicity and carcinogenicity studies.

Qualitative and quantitative evaluation of prostatic histomorphology in rats following chronic treatment with finasteride, a 5-alpha reductase inhibitor.

OBJECTIVE: To determine any potential direct and/or indirect effects of elevated intraprostatic T levels on the prostates of rats chronically (1-2 years) exposed to high doses (160 mg/kg/day) of finasteride, a selective inhibitor of 5-alpha reductase. METHODS: Sprague-Dawley male rats were administered daily finasteride by oral gavage. Prostates from all rats were weighed, fixed in 10% neutral buffered formalin, and processed for light microscopic examination. The volume fractions of the prostatic glandular and stromal compartments were quantitated by morphometric analysis. RESULTS: Administration of finasteride at doses of 20, 40, and 80 mg/kg/day for one year resulted in a significant (P < or = 0.05) decrease in prostatic weight; prostatic atrophy was evident by light microscopy. Morphometric analysis of the prostate showed that chronic finasteride administration resulted in a significant (P < or = 0.001) decrease in the absolute volume of both glandular (-65.2%) and stromal (-57.1%) compartments of the prostate. Furthermore, the total number of epithelial and stromal cells per gland were significantly (P < or = 0.002) decreased in finasteride-treated rats compared with vehicle controls; the magnitude of mean decrease was 69.8 percent and 50.6 percent of controls in epithelial and stromal cells, respectively. In addition, prostates from all two hundred fifty rats in a two-year study were qualitatively evaluated by light microscopy. Administration of finasteride at doses ranging from 2.5 mg/kg/day to 160 mg/kg/day for two years did not result in an increase over the background incidence of prostatic focal hyperplasia or adenoma. No malignant tumors of the prostate were seen in any of the groups. CONCLUSIONS: These studies have demonstrated that the expected pharmacologic effects of finasteride on the prostate are maintained following chronic treatment and that there was no evidence of a direct and/or an indirect effect of elevated intraprostatic T on prostatic morphology in rats.

Recovery of substance P but not somatostatin in the cat spinal cord after unilateral lumbosacral dorsal rhizotomy: a quantitative study.

The dorsal horn of the cat spinal cord contains substance P and somatostatin within nerve endings which arise from cells located in dorsal root ganglia and from cells within the neuraxis. Previous studies from this laboratory have demonstrated that dorsal rhizotomy depletes both peptides from the dorsal horn. However, the changes in the two peptides differ. Substance P is at first severely depleted by dorsal rhizotomy and then recovers in part, whereas somatostatin is diminished less but does not recover. In the present experiments the validity of these conclusions which were based on anatomical observations has been evaluated quantitatively with the use of radioimmunoassay. After a 74% reduction at 10-14 days postoperative, substance P immunoreactivity in the deafferented dorsal horn shows a small, statistically significant recovery by 30 days to 60% of normal values. In contrast, somatostatin is reduced by 46% at 10-14 days but does not return significantly. As previously suggested by immunocytochemistry, dorsal rhizotomy produces no significant decline of either peptide in the ventral horn. The differing response of the two peptides is consistent with the hypothesis that intrinsic spinal substance P-containing neurons increase their projections (or their production of substance P) in the deafferented dorsal horn, but that somatostatin-containing neurons do not. Because synaptic number returns to normal in at least the deafferented lamina II of the cat yet substance P recovers only partially, it is likely that axons which contain transmitters other than substance P or somatostatin also increase the numbers of their terminals in response to dorsal rhizotomy.

Diagnostic testing for coronary artery disease in a large population.

Extensive public health programs are often proposed without a full appreciation of their effects on the target population. There is often a problem of confusing medical care that may be of benefit to an individual with care that may be of benefit to a population: care that may be highly beneficial for a selected patient may be substantially less effective for an unselected population. Exposing a large, asymptomatic population to diagnostic screening for coronary artery disease has cost and risk ramifications far beyond those of discovering people who might benefit from treatment of previously unsuspected disease. A program to screen 20 million people, and treat the most severely affected with coronary artery bypass graft, would cost nearly +9.2 billion. About +21,000 is spent to find each person with disease, while the cost is more than +169,000 for each person surviving surgical therapy. The cost per year of life extended is over +43,000. In excess of 8,000 persons have major complications and nearly 2,000 die from diagnostic testing and therapy. About half of major complications and deaths occur in persons without disease. This model integrating epidemiologic, economic, and decision analytic methods is presented to illustrate the potential use of myriad techniques in addressing population-based medical care and policy options; it does not provide the definitive answer, however, to the complex problem.

Report and summary of the major conclusions from statistics in genotoxicity testing working group from the International Workshop on Genotoxicity Test Procedures (IWGTP), March 1999.

A working group of five statisticians experienced in the use of statistical methods in mutagenicity reviewed aspects of the statistical analysis of genotoxicity test procedures. Issues discussed included methods for integrating biological importance and statistical significance, the relationship of the experimental unit to the experimental design, and the impact of new developments in statistics and computing. Three major recommendations were made relating to the need for: (1) the effective use of statistical advice in designing interlaboratory and intralaboratory investigations; (2) the development of appropriate experimental designs for new assays; and (3) education and training in the use of statistical methodology in mutagenicity testing. Environ. Mol. Mutagen. 35:260-263, 2000 Published 2000 Wiley-Liss, Inc.

Antigen levels and antibody titers after DNA vaccination.

DNA vaccination generates strong cellular and humoral immunity in animal models. The mechanisms by which plasmid DNA uptake and expression after intramuscular injection lead to immune responses are not well understood. In particular, the importance of antigen expression levels on subsequent antibody immune responses has not been established. We found that a chemiluminescent assay for alkaline phosphatase allows measurement of antigen levels of secreted alkaline phosphatase (SEAP) in vivo after intramuscular injection of a wide range of plasmid doses. The mice produced antibodies to the alkaline phosphatase reporter gene and both antigen levels and antibody titers were measured over time. We found that the correlation between initial antigen level and antibody response was high (r = 0.74, p < 0.001) and remained high even after accounting for the dose of plasmid injected (r = 0.61, p < 0.001). The correlation between DNA dose and antibody titer was statistically significant (r = 0.53, p < 0.001) but was reduced to almost zero after we accounted for initial antigen levels.

Replicate flasks are not necessary for in vitro chromosome-aberration assays in CHO cells.

Some recommended protocols for in vitro chromosome-aberration assays call for two flasks per dose group. Use of replicate flasks allows for possible variation in percent aberrant cells (ABR) between flasks. We studied the magnitude of variation between replicate flasks of Chinese hamster ovary (CHO) cells using data from 211 assays from three laboratories, in order to assess the effect on assay sensitivity. Based on all 403 pairs of replicate "control" flasks, there was almost no excess variability between flasks. The standard deviation (SD) was only 4% larger than the value expected purely from sampling cells (P > 0.05). Data from all 366 pairs of replicate "treated" flasks showed that between-flask variation increased with the average percent aberrant cells (P < 0.001). The SD for 60 pairs of flasks with 3.0-7.5% ABR cells was 32% larger than the expected value. However, computer simulations based on these data showed use of replicate flasks has little effect on assay false-positive or true-positive rates. All assays with replicate treated flasks and at least three dose groups including control were re-analyzed as "single-flask" experiments. A "single-flask" experiment was defined by taking both control flasks but only one treated flask per dose. For each assay, all possible single-flask experiments were re-analyzed and the percent with positive results recorded. For most assays, conclusions were the same regardless of which treated flasks were selected, in spite of the fact that these single-flask experiments had only half as many cells scored per active dose group. For a very few assays with marginal results, the conclusion could change depending on which set of flasks was chosen, but these were such borderline results that a repeat assay was required in any case. Repeating the assay is a better way to resolve marginal results than examining replicate flasks. From our re-examination of the experimental data and from the computer simulation, we conclude that, while flask-to-flask variability exists, it has no practical effect on the test outcome, so that use of replicate flasks is not necessary for this assay.

Paradoxical changes in SCE frequencies persistently elevated in vivo, on exposure to a mutagen in vitro.

Lymphocyte cultures from 4 individuals with persistently significantly elevated frequencies of sister-chromatid exchange (SCE) were examined with no treatment, and with 2 concentrations of mitomycin C. In each of the 4 cases, the mean level of SCEs in the untreated lymphocytes exhibited a paradoxical reduction in SCE frequency when exposed to the lower (0.005 microgram/ml) of the two doses of mitomycin C. At the second higher dose of mitomycin C (0.025 microgram/ml) the mean level of SCE/cell exceeded the untreated mean. When the distributions of SCE/cell were examined it appeared that the untreated cultures had two or more populations of cells; one was in the normal SCE frequency range, while the second population was in an elevated SCE frequency range. The paradoxical reduction in SCE frequency was apparently due to elimination of, or mitotic inhibition of cells in the highest range of SCE frequency, while a small elevation in SCEs was initiated in the cells with a normal SCE frequency. Thus, mean levels of SCE/cell can be misleading. This data suggests that new exposure to the same or a different genotoxic agent might possibly result in a misleading lowering of the mean SCE frequency.

Sister-chromatid exchange (SCE) report on control subjects in a study of occupationally exposed workers.

The range and distribution of Sister-Chromatid Exchange (SCE) scores in 479 control persons were determined. All SCE readings were performed in a single laboratory according to the same protocol and regularly checked by referee readers to assure consistency. A mean SCE per cell value of 9.9 and a 95th percentile of 13.4 were established for this study sample. The range of SCE scores across all non-exposed individuals tested was 5.0-17.5 SCE per cell. Differences in SCE scores were associated with reader, smoking, sex, and, to a small extent, age. Individual test results showed reasonable consistency across the entire control group, but, as with most clinical measurements, care should be taken to avoid placing too much emphasis on a single test result when communicating with an individual. This report on the largest control group studied to date provides necessary normative data for further SCE investigations in occupational settings.

Sister-chromatid exchanges in association with occupational exposure to ethylene oxide.

Sister-chromatid exchange (SCE) frequencies in employees potentially exposed to ethylene oxide (ETO) were compared with those in unexposed control groups. Three worksites where the previous environmental control of ETO was known to have differed were chosen. Within these worksites, subjects were categorized into high potential exposed, low potential exposed and control groups. An additional community control group was obtained. Blood samples for chromosome studies of peripheral lymphocytes were drawn at several time points over a period of 24 months. The effects on SCE of age, sex, smoking habits and reader variation were considered. Worksites I, II and III, respectively, represented increasing levels of exposure. At Worksite III large differences among groups persisted over 24 months. At Worksite II, the SCEs in the high potential exposed workers were higher than those in the other groups. At no time was the low potential exposed group at Worksite II statistically significantly higher in mean SCE than the worksite controls. No consistent differences among groups were noted in Worksite I.