Food rationing affects dietary selenium bioaccumulation and life cycle performance in the mayfly Centroptilum triangulifer.

Selenium effects in nature are mediated by the relatively large bioconcentration of aqueous Se by primary producers and smaller, yet critical, dietary transfers to primary consumers. These basal processes are then propagated through food webs to higher trophic levels. Here we quantified the movement of dissolved Se (as selenite) to periphyton, and used the resultant periphyton as a food source for conducting full life-cycle dietary Se exposures to the mayfly Centroptilum triangulifer. Periphyton bioconcentrated Se ~2,200-fold from solution in a log-linear fashion over dissolved Se concentrations ranging from 1.1 to 23.1 µg L(-1). We examined the influence of two feeding ration levels (1x and 2x) on trophic transfer, tissue Se concentrations, maternal transfer, and functional endpoints of mayfly performance. Mayflies fed a lesser ration (1x) displayed greater trophic transfer factors (mean TTF, 2.8 ± 0.4) than mayflies fed 2x rations (mean TTF, 1.1 ± 0.3). In 1x exposures, mayflies exhibited significant (p < 0.05) reductions in survivorship and total body mass at dietary [Se] ≥ 11.9 µg g(-1), reduced total fecundity at ≥ 4.2 µg g(-1), and delayed development at ≥ 27.2 µg g(-1). Mayflies fed a greater ration (2x) displayed reduced tissue Se concentrations (apparently via growth dilution) relative to 1x mayflies, with no significant effects on performance. These results suggest that the influence of Se on mayfly performance in nature may be tied to food resource availability and quality. Furthermore, nutritional status is an important consideration when applying laboratory derived estimates of toxicity to risk assessments for wild populations.

Correlation analysis of gene expression and clinical chemistry to identify biomarkers of skeletal myopathy in mice treated with PPAR agonist GW610742X.

Data from individual animals were used to identify genes in mouse skeletal muscle whose expression correlated with a known serum marker of skeletal myopathy, creatine kinase activity (CK), after treatment with a peroxisome proliferator-activated receptors (PPAR) agonist, GW610742X. Six genes had correlation coefficients of >or=0.90: Mt1a (metallothionein 1a), Rrad (Ras-related associated with diabetes), Ankrd1 (ankyrin repeat domain 1), Stat3 (signal transducer and activator of transcription 3), Socs3 (suppressor of cytokine signalling 3) and Mid1ip1 (Mid1 interacting protein 1). The physiological function of these genes provides potentially useful information relating to the mechanism of PPAR-induced skeletal myopathy, with oxidative stress and disruption of glycolysis most closely associated with myopathic damage. Some of the muscle genes most highly correlated with serum CK in mice also appear to be good indicators of PPAR-induced myopathy in rat skeletal muscle, demonstrating the translational potential of this approach. This study clearly shows the utility of using correlation analysis as a simple tool for identifying novel biomarkers and investigating mechanisms of toxicity.

Visualization and quantitation of peroxisomes using fluorescent nanocrystals: treatment of rats and monkeys with fibrates and detection in the liver.

Peroxisome proliferation in the liver is a well-documented response that occurs in some species upon treatment with hypolipidemic drugs, such as fibrates. Typically, liver peroxisome proliferation has been estimated by direct counting via electron microscopy, as well as by gene expression, enzyme activity, and immunolabeling. We have developed a novel method for the immunofluorescent labeling of peroxisomes, using an antibody to the 70-kDa peroxisomal membrane protein (PMP70) coupled with fluorescent nanocrystals, Quantum Dots. This method is applicable to standard formalin-fixed, paraffin-embedded tissues. Using this technique, a dose-dependent increase in PMP70 labeling was evident in formalin-fixed liver sections from fenofibrate-treated rats. In formalin-fixed liver sections from cynomolgus monkeys given ciprofibrate, quantitative image analysis showed a statistically significant increase in PMP70 labeling compared to control; the increase in hepatic PMP70 protein levels was corroborated by immunoblotting using total liver protein. An increase in hepatic peroxisome number in ciprofibrate-treated monkeys was confirmed by electron microscopy. An advantage of the Quantum Dot/PMP70 method is that a single common protocol can be used to label peroxisomes from several different species, and many of the common problems that arise with immunolabeling, such as fading and low signal strength, are eliminated.

Elevated HPRT mutation frequencies in aflatoxin-exposed residents of daxin, Qidong county, People's Republic of China.

Molecular biomarkers are becoming increasingly important tools to identify people who are at highest risk of developing cancer. For many years we have been studying residents of Qidong County, People's Republic of China, to examine the combined impact of aflatoxin exposure with other risk factors as contributors to the high liver cancer incidence rates in this region. This study was conducted to determine the effects of aflatoxin exposure, as measured by serum aflatoxin-albumin adduct levels, on somatic mutation frequency in the human hypoxanthine guanine phosphoribosyl transferase gene (HPRT). Subjects were assigned as low or high according to a dichotomization around the population mean of aflatoxin-albumin adducts. HPRT mutant frequency was determined in individuals by a T cell clonal assay and the samples were categorized as low or high according to mean values. Separate analyses were also conducted for the small set of hepatitis B virus surface antigen (HBsAg)-positive and the larger set of HBsAg-negative individuals, known risk factors for liver cancer. An odds ratio of 19.3 (95% confidence interval 2.0, 183) was demonstrated for a high HPRT mutation frequency in individuals with high aflatoxin exposure compared with those with low aflatoxin exposure. This association indicates that aflatoxin-induced DNA damage in T lymphocytes, assessed using the validated surrogate albumin adduct markers, leads to increased mutations reflected as elevated HPRT gene mutations. This cross-sectional study suggests the potential use of mutation frequency of the HPRT gene as a long-term biomarker of aflatoxin exposure in high risk populations.

Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are found.

A novel bacterial reversion and forward mutation assay based on green fluorescent protein.

We report the first use of green fluorescent protein (GFP) for mutation detection. We have constructed a plasmid-based bacterial system whereby mutated cells fluoresce and non-mutated cells do not fluoresce. Fluorescence is monitored using a simple hand-help UV lamp; no additional cofactors or manipulations are necessary. To develop a reversion system, we introduced a +1 DNA frameshift mutation in the coding region of GFP and the resulting protein is not fluorescent in Escherichia coli. Treatment of bacteria containing the +1 frameshift vector with ICR-191 yields fluorescent colonies, indicating that reversion to the wild-type sequence has occurred. Site-directed mutagenesis was used to insert an additional cytosine into a native CCC sequence in the coding region of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A dose-related increase in fluorescent colonies was observed when the bacteria were treated with ICR-191, an agent that induces primarily frameshift mutations. The highest dose of ICR-191 tested, 16 microg/ml, produced a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate experiments. The reversion system did not respond to MNNG, an agent that produces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose PBAD promoter; in the absence of arabinose, GFP expression is repressed and no fluorescent colonies are observed. When cells were treated with MNNG or ENNG, a dose-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-repressed the promoter. Treating bacteria with 100 microg/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 40 x 10-5 in duplicate experiments. Treating bacteria with 150 microg/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment.

Databases and software for the analysis of mutations in the human p53 gene, human hprt gene and both the lacI and lacZ gene in transgenic rodents.

We have created databases and software applications for the analysis of DNA mutations at the human p53 gene, the human hprt gene and both the rodent transgenic lacI and lacZ loci. The databases themselves are stand-alone dBASE files and the software for analysis of the databases runs on IBM-compatible computers with Microsoft Windows. Each database has a separate software analysis program. The software created for these databases permit the filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web. Open the following home page with a Web Browser: http://sunsite.unc.edu/dnam/mainpage. html . Alternatively, the databases and programs are available via public FTP from: anonymous@sunsite.unc.edu. There is no password required to enter the system. The databases and software are found beneath the subdirectory: pub/academic/biology/dna-mutations. Two other programs are available at the site, a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

The Ames miniscreen assay: volatility of sodium azide can cause an increase in the reversion frequencies of adjacent, untreated wells.

Sodium azide, when added to wells adjacent to untreated wells, caused an increase in the reversion rate of Salmonella typhimurium TA100 in a 12-well plate format. Increases in the reversion frequency in adjacent, untreated wells were observed when a single well on the plate was treated with as little as 1 microg of sodium azide. This effect is probably caused by the hydrolysis of sodium azide to form hydrazoic acid. Hydrazoic acid has a boiling point of 37 degrees C and, thus, would become a diffusible gas during the incubation of the plates. Our findings suggest that a diffusible gas is present and that this gas has the ability to contaminate nearby wells when using the multiwell version of the Ames assay. Furthermore, it may be prudent to isolate all positive controls and negative controls on separate plates with no test material since a volatile test material could produce spurious results in the Ames miniscreen.

Databases and software for the analysis of mutations in the human p53 gene, the human hprt gene and both the lacI and lacZ gene in transgenic rodents.

We have created databases and software applications for the analysis of DNA mutations at the humanp53gene, the humanhprtgene and both the rodent transgeniclacIandlacZlocus. The databases themselves are stand-alone dBASE files and the software for analysis of the databases runs on IBM-compatible computers. Each database has a separate software analysis program. The software created for these databases permit the filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web (WWW). Open the following home page with a Web Browser: http://sunsite.unc.edu/dnam/mainpage.ht ml . Alternatively, the databases and programs are available via public FTP from: anonymous@sunsite.unc.edu . There is no password required to enter the system. The databases and software are found beneath the subdirectory: pub/academic/biology/dna-mutations. Two other programs are available at the site-a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

The Ames test: the two-fold rule revisited.

Mutagenicity in the Ames assay is evaluated by comparing the number of revertants observed in treated cultures to those in untreated cultures. Often, some form of the '2-fold rule' is employed, whereby a compound is judged mutagenic if a 2-fold or greater increase is seen in a treated culture. In order to understand the underpinnings of this approach, we study some of its statistical properties. We assume that the number of revertants on any plate from a given two-group experiment follows a Poisson distribution and we address the following questions: (1) what is the false-positive error probability of observing at least a doubling of the number of colonies from the control to the treatment group?; (2) if a given mean number of colonies is postulated for a control group, what number of colonies above the observed control mean provides a false-positive rate of 5%? We also present results for question 1 in the case where the number of revertants follows a negative binomial distribution.

Relational database model for DNA mutations and a software program for implementation of the model.

A relational database model for describing DNA mutations is presented. The model was developed in conjunction with the human hprt database and was successful in representing over 1800 hprt mutations. Mutants showing aberrant mRNA splicing can be adequately described using the model, as well as mutants showing more than one mutation. The basic aspects of the relational model should be applicable to mutations in a variety of genes. A data entry program developed using Microsoft Access 2.0 is also described that implements the relational model. The data entry program ensures that relational integrity is maintained between the tables and automatically generates key fields as needed. The program also has the ability to convert between the various numbering schemes that are used to describe base pair location in the hprt gene. The program and source code are placed in the public domain so that other experimenters can adapt the program for use with other genes.

Database and software for the analysis of mutations at the lacI gene in both transgenic rodents and bacteria.

The use of transgenic rodents for the study of genetic toxicology has increased dramatically in the past several years. A great deal of the recent work has employed the lacI locus in transgenic mice. In addition to the transgenic data, a substantial amount of information exists regarding mutation of the lacI gene in bacteria. In an effort to centralize the information regarding mutations in the lacI gene in both rodents and bacteria, we have created a computerized database that contains information about DNA sequence alterations on about 500 mutations in transgenic rodents and 8,000 mutations in bacteria. We have also produced a software package for the analysis of the lacI database. Routines have been developed for the analysis of single base substitutions, including programs to (i) determine if two mutational spectra are different; (ii) determine if mutations show a DNA strand bias; (iii) determine the frequency of transitions and transversions; (iv) display the number and kind of mutations observed at each base in the coding region; (v) perform nearest neighbor analysis; and (vi) display mutable amino acids in the lacI protein. The software runs only on IBM-compatible machines running Microsoft Windows. The software and lacI database are freely available via the internet (http:/(/)sunsite.unc.edu/dnam/mainpage.++ +html).

Database and software for the analysis of mutations at the lacZ locus in transgenic rodents.

The use of transgenic rodents is becoming increasingly widespread in genetic toxicology. In an effort to centralize and standardize the information regarding mutations in rodents bearing the lacZ transgene, we have created a computerized database that contains published information about DNA sequence alterations on over 100 mutants. Information on the literature citation, mutagenic conditions, organs from specific animals, mutation frequency in each organ, specific mutation, amino acid change, and other data are provided for each mutant. We have also produced a software package for the analysis of the lacZ database. Routines have been developed for the analysis of single base substitutions, including programs to 1) determine whether two mutational spectra are statistically different, 2) determine whether mutations show a DNA strand bias, 3) determine the frequency of transitions and transversions, 4) display the number and kind of mutations observed at each base in the coding region, 5) perform nearest-neighbor analysis, and 6) display mutable amino acids in the lacZ protein. The software runs only on IBM-compatible machines running Microsoft Windows. The software and lacZ database are freely available via the Internet (http:@sunsite.unc.edu/dnam/mainpage.ht ml). These programs simplify the analysis of the rapidly increasing information about lacZ mutation. The programs permit the facile comparison between different lacZ data sets as well as the identification of mutational patterns that may be of importance to experimenters studying the mechanisms of mutation and mutational spectra in transgenic animals.

Databases and software for the analysis of mutations in the human p53 gene, the human hprt gene and the lacZ gene in transgenic rodents.

We have created databases and software applications for the analysis of DNA mutations in the human p53 gene, the human hprt gene and the rodent transgenic lacZ locus. The databases themselves are stand-alone dBase files and the software for analysis of the databases runs on IBM- compatible computers. The software created for these databases permits filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web (WWW). Open home page http://sunsite.unc.edu/dnam/mainpage.ht ml with a WWW browser. Alternatively, the databases and programs are available via public ftp from anonymous@sunsite.unc.edu. There is no password required to enter the system. The databases and software are found in subdirectory pub/academic/biology/dna-mutations. Two other programs are available at the WWW site, a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

Computer program for the analysis of mutational spectra: application to p53 mutations.

Mutations in the p53 oncogene are extremely common in human cancers, and environmental exposure to mutagenic agents may play a role in the frequency and nature of the mutations. Differences in the patterns of p53 mutations have been observed for different tumor types. It is not trivial to determine if the differences observed in two mutational spectra are statistically significant. To this end, we present a computer program for comparison of two mutational spectra. The program runs on IBM-compatible personal computers and is freely available. The input for the program is a text file containing the number and nature of mutations observed in the two spectra. The output of the program is a P value, which indicates the probability that the two spectra are drawn from the same population. To demonstrate the program, the mutational spectra of single base substitutions in the p53 gene are compared in (i) bladder cancers from smokers and non-smokers, (ii) small-cell lung cancers, non-small-cell lung cancers and colon cancers and (iii) hepatocellular carcinomas from high- and low-aflatoxin exposure groups. p53 mutations differ in several important aspects from a typical mutational spectra experiment, where a homogeneous population of cells is treated with a specific mutagen and mutations at a specific locus are recovered by phenotypic selection. The means by which p53 mutations are recognized is by the appearance of a cancer, and this phenotype is very complex and varied.

Database and software for the analysis of mutations at the human hprt gene.

A computerized database containing DNA sequence information regarding human HPRT mutants has been created. The database itself is in the dBASE format and contains information on about 1500 mutants. In addition, an IBM PC compatible software package to analyze the information in the database has been developed. Both the database and software are freely available via the Internet.

Database and software for the analysis of mutations at the human p53 gene.

A computerized database containing DNA sequence information regarding human p53 mutants has been created. The database itself is in the dBASE format and contains information on nearly 3000 mutants. In addition, an IBM PC compatible software package to analyze the information in the database has been developed. Both the database and software are freely available via the Internet.

In vitro mutational spectrum of aflatoxin B1 in the human hypoxanthine guanine phosphoribosyltransferase gene.

The in vitro mutational spectrum of aflatoxin B1 (AFB1) in exon 3 of the human hypoxanthine guanine phosphoribosyltransferase gene in B-lymphoblasts was examined by a combination of polymerase chain reaction and denaturing gradient gel electrophoresis. The cell line used in this study contained an expression vector that produced high levels of human cytochrome P450 CYP1A1. CYP1A1 metabolizes AFB1 to form an epoxide intermediate which can react with DNA. About 1200 independent mutants were induced at the hypoxanthine guanine phosphoribosyltransferase locus by AFB1 and were selected en masse by addition of 6-thioguanine to the bulk culture. Two independent cultures were treated with AFB1. Polymerase chain reaction was used to amplify exon 3 from the complex mutant population, and denaturing gradient gel electrophoresis was used to separate wild-type DNA sequences from mutant sequences. Mutational hotspots were visible as discrete bands on the denaturing gradient gel. Scanning densitometry was used to determine the fraction of the complex population that was represented in each non-wild-type band. The bands containing the mutations were excised from the denaturing gradient gel and sequenced. In this way, the nature and frequency of mutational hotspots in a population of > 1000 mutants were determined. AFB1 produced one strong mutational hotspot in exon 3. Between 10 and 17% of the AFB1-induced mutants contained a single GC-->TA base substitution at base pair 209. This hotspot occurred in a GGGGGG sequence (the mutated base is underlined). This mutation was observed reproducibly in two independently treated cultures. Several other mutations were observed in only one culture but at a lower frequency. Our results are the first report of the mutational spectrum of AFB1 in a native human gene.

Database and software for the analysis of mutations in the human p53 gene.

Mutations of the human p53 gene are of importance in the development of cancer. Perhaps 50% of all human cancers contain a mutation in the p53 oncogene and many laboratories are investigating mutations at this locus. In an effort to centralize and standardize the information regarding human p53 mutations, we have created a computerized database that contains information about DNA sequence alterations for > 3000 p53 mutants. Information on the cancer type, the origin of the cells, the specific mutation, the amino acid change, the literature citation, and other data are provided for each mutant. We have also produced a software package for the analysis of the p53 database. Routines have been developed for the analysis of single-base substitutions, including programs to (a) determine whether two mutational spectra are different, (b) display the number of mutations and mutable sites in each exon, (c) determine whether mutations show a DNA strand bias, (d) determine the frequency of transitions and transversions, (e) display the number and kind of mutations observed at each base in the coding region, (f) perform nearest neighbor analysis, and (g) display mutable amino acids in the p53 protein. The software runs only on IBM-compatible machines with MS-DOS. The software and p53 database are freely available via the Internet, using the remote file transfer protocol. These programs simplify the analysis of the rapidly increasing body of information about p53 mutations. The programs permit facile comparison between different p53 data sets, as well as the identification of mutational patterns that may be of importance to experimenters studying the mechanisms of mutation and the etiology of cancers.

Software for the analysis of mutations at the human hprt gene.

Mutations at the human hypoxanthine-guanine phosphoribosyl transferase gene (hprt) are currently of great interest because mutations at this locus are being used as a biomonitor of human mutagenic exposure. Not only can somatic hprt mutants arising in vivo in humans be recovered and sequenced, but there is also a considerable body of information about the in vitro mutational spectra of different carcinogens at this locus. Previously, we reported the creation of a computerized database containing DNA-sequence information on human hprt mutants (Cariello et al. (1992) Environ. Mol. Mutagen., 20, 81-83). In the present manuscript, software for the analysis of mutations in the hprt database is described. Numerous routines have been developed for the analysis of single-base substitutions, including programs to (i) determine if two mutational spectra are different, (ii) display the number of mutations and mutable sites in each exon, (iii) determine if mutations show a DNA-strand bias, (iv) determine the frequency of transitions and transversions, (v) display the number and kind of mutations observed at each base in the coding region, (vi) perform nearest-neighbor analysis and (vii) display mutable amino acids in the hprt protein. The software runs only on IBM-compatible machines with MS-DOS. The software and hprt database is freely available via the INTERNET using remote file-transfer protocol. These programs simplify the analysis of the rapidly increasing information about hprt mutation. The programs permit the facile comparison between in vitro and in vivo data, as well as the identification of mutational patterns that may be of importance to experimenters using hprt as a biomonitor and and of importance to researchers studying mechanisms of mutation.