Pancreatic inflammation and increased islet macrophages in insulin-resistant juvenile primates.

Chronic high caloric intake has contributed to the increased prevalence of pediatric obesity and related morbidities. Most overweight or obese children, however, do not present with frank metabolic disease but rather insulin resistance or subclinical precursors. The innate immune system plays a role in the pathophysiology of type 2 diabetes but how it contributes to early metabolic dysfunction in children on chronic high-fat diet (HFD) is unclear. We hypothesize that such inflammation is present in the pancreas of children and is associated with early insulin resistance. We used nonhuman primate (NHP) juveniles exposed to chronic HFD as a model of early pediatric metabolic disease to demonstrate increased pancreatic inflammatory markers before the onset of significant obesity or glucose dysregulation. Pancreata from 13-month-old Japanese macaques exposed to a HFD from in utero to necropsy were analyzed for expression of cytokines and islet-associated macrophages. Parameters from an intravenous glucose tolerance test were correlated with cytokine expression. Before significant glucose dysregulation, the HFD cohort had a twofold increase in interleukin 6 (IL6), associated with decreased first-phase insulin response and a sexually dimorphic (male) increase in IL1ß correlating with increased fasting glucose levels. The number of islet-associated macrophages was also increased. Pancreata from juvenile NHP exposed to HFD have increased inflammatory markers and evidence of innate immune infiltration before the onset of significant obesity or glucose dysregulation. Given the parallel development of metabolic disease between humans and NHPs, these findings have strong relevance to the early metabolic disease driven by a chronic HFD in children.

Chromosome aberration assays in Pisum for the study of environmental mutagens.

From a literature survey, 117 chemicals are tabulated that have been assayed in 179 assays for their clastogenic effects in Pisum. Of the 117 chemicals that have been assayed, 65 are reported at giving a positive reaction (i.e. causing chromosome aberrations), 30 positive with a dose response, five borderline positive. Seventeen chemicals gave a negative response. Eighty-one percent of the chemicals gave a definite positive response. A c-mitotic effect was detected from treatment with 17 chemicals. In addition to the above tabulation of chemicals, 39 chemicals have been reported with an antimitotic effect. Thirteen assays have been recorded for five types of radiation, which with the exception of ultrasound reacted positively. The results of assays with 38 chemicals and/or radiations in combined treatments, as well as 15 chemicals and three types of radiations that induce somatic mutations are tabulated. The Pisum sativum (2n=14) bioassay has been shown to be a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis for somatic mutations induced by chemicals, radiations, and environmental pollutants. For some chemicals, the Pisum assay is not as sensitive in assessing clastogenicity as the Allium assay, although this should be considered in relative terms. Pisum fulvum (2n=14) has been used in clastogenic studies also, but to a much lesser extent.

Higher plant assays for the detection of chromosomal aberrations and gene mutations-a brief historical background on their use for screening and monitoring environmental chemicals.

Higher plants are recognized as excellent indicators of cytogenetic and mutagenic effects of environmental chemicals and are applicable for the detection of environmental mutagens both indoor and outdoor. They are highly reliable bioassays with a high sensitivity for monitoring and testing for genotoxins. A brief review of major steps in the development of higher plant genotoxic assays is given.

Chromosome aberration assays in Crepis for the study of environmental mutagens.

Crepis capillaris (2n=6) is an excellent plant for the assay of chromosome aberrations after chemical treatment. C. tectorum (2n=8) has been used also in mutagenic studies, but to a much lesser extent. A protocol has been given for using root tips to study the cytological endpoints, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. Meiotic endpoints have not been used in C. capillaris for testing potential chemical mutagens but should be considered, especially a meiotic micronucleus assay. From a literature survey, 81 chemicals are tabulated that have been assayed in 162 Crepis assays for their clastogenic effects. Of the 162 assays that have been carried out, 40 are reported at giving a positive reaction (i.e., causing chromosome aberrations), 97 positive and with a dose response, 7 borderline positive, and 17 negative. Eighty-five percent of the chemicals gave a definite positive response. Assays for one chemical gave contrary results, and were not included in the above tabulation. The Crepis bioassay has been shown to be an excellent plant bioassay for assessing chromosome damage induced by chemicals and environmental pollutants.

The present status of higher plant bioassays for the detection of environmental mutagens.

Higher plants provide valuable genetic assay systems for screening and monitoring environmental pollutants. They are now recognized as excellent indicators of cytogenetic and mutagenic effects of environmental chemicals and are applicable for the detection of environmental mutagens both indoor and outdoor. Comparisons between plant and nonplant genetic assay systems indicate that higher plant genetic assays have a high sensitivity (i.e. few false negatives). Two assays which are considered ideal for in situ monitoring and testing of airborne and aqueous mutagenic agents are the Tradescantia stamen hair assay for mutations and the Tradescantia micronucleus assay for chromosome aberrations. Both assays can be used for in vivo and in vitro testing. Other higher plant genotoxicity assays which have a large number of genetic markers and/or data base and are also highly suitable for testing for genotoxic agents include Arabidopsis thaliana, Allium cepa, Hordeum vulgare, Vicia faba, and Zea mays. Since higher plant systems are now recognized as excellent indicators of the cytotoxic, cytogenetic, and mutagenic effects of environmental chemicals and have unique advantages for in situ monitoring and screening it is recommended that higher plant systems be accepted by regulatory authorities as an alternative first-tier assay system for the detection of possible genetic damage resulting from pollution or the use of environmental chemicals. The results from higher plant genetic assays could make a significant contribution in protecting the public from agents that can cause mutation and cancer. The advantages possessed by higher plant genetic assays, which are inexpensive and easy to handle, make them ideal for use by scientists in developing countries.

Environmental monitoring for genotoxicity with plant systems. An introduction and study design.

Under the sponsorship of the International Programme on Chemical Safety (IPCS), 17 laboratories from diverse regions of the world participated in evaluating the utility of four plant bioassays for detecting genetic hazards of environmental chemicals. The bioassays included in this collaborative study were: Arabidopsis thaliana embryo and chlorophyll assay and Tradescantia stamen hair assay, Tradescantia paludosa micronucleus assay and Vicia faba root tip assay. Four to six laboratories participated in the performance of each of the bioassays. All laboratories participating in a particular bioassay were supplied with uniform plant material as well as standardized protocol. Five direct acting water soluble test chemicals, i.e. maleic hydrazide, methyl nitrosourea, ethyl methanesulfonate, sodium azide and azidoglycerol, were selected for this study. The study was designed to be completed in three phases. Ethyl methanesulfonate was used as a positive control and has already been reported earlier (Sandhu et al., 1991). The data from the remaining four chemicals used for the evaluation of four plant test systems in the first phase of the collaborative study are reported in this issue.

Comparative mutagenicity of chemicals selected for test in the International Program on Chemical Safety's collaborative study on plant systems for the detection of environmental mutagens.

A review has been made for the four compounds (maleic hydrazide, methyl nitrosourea, sodium azide, azidoglycerol) tested in the International Program on Chemical Safety's collaborative study on plant systems. Maleic hydrazide (MH) is a weak cytotoxic/mutagenic chemical in mammalian tissues and is classified as a class 4 chemical. In contrast, with few exceptions such as Arabidopsis, MH is a potent mutagen/clastogen in plant systems. The difference in its response between plant and animal tissue is likely due to differences in the way MH is metabolized. MH appears to be noncarcinogenic and has been given a negative NCI/NTP carcinogen rating. Methyl nitrosourea (MNU) is a toxic, mutagenic, radiomimetic, carcinogenic, and teratogenic chemical. It has been shown to be a mutagen in bacteria, fungi, Drosophila, higher plants, and animal cells both in vitro and in vivo. MNU is a clastogen in both animal and human cell cultures, plant root tips and cell cultures inducing both chromosome and chromatid aberrations as well as sister-chromatid exchanges. Carcinogenicity has been confirmed in numerous studies and involves the nervous system, intestine, kidney, stomach, bladder and uterus, in the rat, mouse, and hamster. MNU produces stage-specific teratogenic effects and also interferes with embryonic development. The experimental evidence that strongly indicates the mutagenic effects of MNU underlines the possible hazard of this compound to human beings. The experimental evidence for the stringent handling of this compound is clear. Sodium azide (NaN3) is cytotoxic in several animal and plant systems and functions by inhibiting protein synthesis and replicative DNA synthesis at low dosages. It is mutagenic in bacteria, higher plants and human cells and has been used as a positive control in some systems. In general, tests for clastogenicity have been negative or weakly positive. No evidence of carcinogenicity has been reported in a 2-year study seeking carcinogenic activity in male and female rats. Its advantages in comparison to other efficient mutagens are claimed to be a high production of gene mutations accompanied by a low frequency of chromosomal rearrangements and safer handling because of its nonclastogenic and noncarcinogenic action on humans. Azidoglycerol (AG) is a very potent mutagen in bacteria, yeast and higher plants including Arabidopsis and Tradescantia; however, it only slightly enhances the frequencies of recessive lethals in Drosophila. AG is at best a weak clastogen and is without effect in inducing chromosomal aberrations and SCEs in human peripheral lymphocytes in vitro. In microbial and plant systems, AG is considerably more potent than sodium azide in the maximal frequencies of mutation induced. In particular, in Saccharomyces cerevisae, AG is 3000-fold more mutagenic than sodium azide. Its carcinogenic and teratogenic properties are unknown.

Environmental monitoring for genotoxicity with plant systems. Results and recommendations.

In the first phase of a collaborative study by the International Programme on Chemical Safety (IPCS), four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), methyl nitrosourea (MNU), sodium azide (NaN3) and maleic hydrazide (MH), and ethyl methanesulfonate (EMS) as a positive control were tested in four plant bioassays, namely the Arabidopsis embryo and chlorophyll mutation assay, the Tradescantia stamen hair assay (Trad-SH assay), the Tradescantia micronucleus assay (Trad-MCN), and the Vicia faba root tip assay. Seventeen laboratories from diverse regions of the world participated with four to six laboratories each using one plant assay. For the Arabidopsis assay, laboratories were in agreement with MNU and AG giving positive responses and NaN3 giving a negative response. With the exception of one laboratory which reported MH as weakly mutagenic, no mutagenic response was reported for MH by the other laboratories. For the Vicia faba assay, all laboratories reported a positive response for MNU, AG, and MH, whereas two of the six laboratories reported a negative response for NaN3. For the Trad-SH assay, MH was reported as giving a positive response and a positive response was also observed for MNU with the exception of one laboratory. NaN3, which exhibited a relatively high degree of toxicity, elicited a positive response in three of the five laboratories. AG was found positive in only one of the two laboratories which tested this chemical. For the Trad-MCN assay, MNU and MH were reported as positive by all laboratories, while four out of five laboratories reported NaN3 to be positive. Only one of three laboratories reported AG to be positive. The major sources of variability were identified and considered to be in the same range as found in similar studies on other test systems. Recommendations were made for minor changes in methodology and for initiating the second phase of this study.

Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD).

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.

The use of Tradescantia and Vicia faba bioassays for the in situ detection of mutagens in an aquatic environment.

Tests have shown plant bioassays to be excellent for mutagenicity studies. Most studies with plant bioassays, however, have been carried out either in the laboratory, or if, in situ, as monitors of atmospheric contaminants. The primary purpose of this study was to assess the utility of in situ plant mutagenicity bioassays in monitoring water contaminants. The assay systems tested were the Tradescantia stamen hair and micronucleus assays for the detection of gene mutations and chromosomal aberrations respectively, and the Vicia faba bioassay system which detects chromosomal aberrations in root tips. The assays were used to test the effluent from a pulp and paper mill located on the north shore of Lake Superior. Assays were performed in a creek containing raw effluent and in the bay of Lake Superior into which the creek emptied. All in situ treatments were carried out for 24 h. The effluent from the creek was heavy with pulp and debris which coated the plant cuttings and the Vicia faba seedlings and may have restricted the uptake from the effluent. In the creek, at test sites 11.5 km from the source, the effluent was toxic to the Vicia faba roots as evidenced by a reduction in the mitotic index. The data for the Tradescantia stamen hair assay in the creek were equivocal. The cuttings from the creek test sites and the air and water control sites appeared to have undergone a physiological delay. Within a day or two after the return to the laboratory, that is 6-8 days after testing, flowering almost ceased and did not fully resume until about day 35. This reduction in flowering was particularly severe with the cuttings from the effluent and air control sites, making it very difficult to interpret the results. In contrast, the Tradescantia micronucleus and Vicia faba chromosomal aberration data were unequivocal; each produced positive responses at both test sites relative to the air and water controls. The results obtained for the bay sites with all 3 assays were in agreement. In that section of the bay visibly contaminated by the creek effluent, increases in stamen hair mutants, micronuclei, and chromosome aberrations were measured. In general, there was a considerable reduction in the number of mutant events observed for the water samples brought back from the test sites and tested in the laboratory.(ABSTRACT TRUNCATED AT 400 WORDS)

The development of sulfonylurea herbicide-resistant birdsfoot trefoil (Lotus corniculatus) plants from in vitro selection.

Herbicide-resistant lines of birdsfoot trefoil (Lotus corniculatus L. cv 'Leo') were isolated after sequential selection at the callus, shoot, and whole plant levels to the sulfonylurea (SU) herbicide Harmony {DPX-M6316; 3-[[[(4-methoxy-6methyl-1,3,5, triazine-2-yl) amino] carbonyl] amino] sulfonyl-2-thiophenecarboxylate}. In field and growth chamber tests the Harmony regenerant lines displayed an increased tolerance as compared to control plants from tissue culture and controls grown from seed. Results of evaluation of callus cultures of regenerated mutant lines signify stability of the resistance. Outcrossed seeds collected from field trials, and tested in vitro for herbicide resistance, indicate that the trait is heritable and that resistance may be due to reduced sensitivity of acetolactate synthase to SU inhibition. Genetically stable herbicide-resistant lines of birdsfoot trefoil were successfully isolated using in vitro selection.

Motion sickness severity and physiological correlates during repeated exposures to a rotating optokinetic drum.

Fifty-two subjects were exposed to a rotating optokinetic drum. Ten of these subjects who became motion sick during the first session completed two additional sessions. Subjects' symptoms of motion sickness, perception of self-motion, electrogastrograms (EGGs), heart rate, mean successive differences of R-R intervals (RRI), and skin conductance were recorded for each session. The results from the first session indicated that the development of motion sickness was accompanied by increased EGG 4-9 cpm activity (gastric tachyarrhythmia), decreased mean successive differences of RRI, increased skin conductance levels, and increased self-motion perception. The results from the subjects who had three repeated sessions showed that 4-9 cpm EGG activity, skin conductance levels, perception of self-motion, and symptoms of motion sickness all increased significantly during the drum rotation period of the first session, but increased significantly less during the following sessions. Mean successive differences of RRI decreased significantly during the drum rotation period for the first session, but decreased significantly less during the following sessions. In conclusion, we have demonstrated that the development of motion sickness is accompanied by an increase in gastric tachyarrhythmia, and an increase in sympathetic activity and a decrease in parasympathetic activity, and that adaptation to motion sickness is accompanied by the recovery of autonomic nervous system balance.

An isoenzyme study in the genus Lotus (Fabaceae) : Segregation of isoenzyme alleles in synthetic allo- and autotetraploids, and in L. corniculatus.

Segregation of the cytosolic Pgi2 locus was studied among progeny of the synthetic allotetraploid (L. japonicus × L. alpinus)(2), the synthetic autotetraploid (L. alpinus)(2), and the cultivated tetraploid species L. corniculatus L. Evidence of an original diploid duplication found within the interspecific hybrid L. japonicus × L. alpinus was also found within the synthetic allotetraploid (quadruplication of loci). Evidence suggesting quadruplication of loci was also found in the tetraploid L. corniculatus, but not in the synthetic autotetraploid (L. alpinus)(2). It is suggested that the original duplication resulted from unequal crossing-over between homoeologues and that it provides evidence that L. corniculatus is a segmental allotetraploid. Quadruplication of loci in L. corniculatus could explain previously reported distorted tetrasomic ratios for segregation of qualitative characters in this species.

An isoenzyme study in the genus Lotus (Fabaceae). Experimental protocols and genetic basis of electrophoretic phenotype.

An isoenzyme survey of some taxa in the genus Lotus (Fabaceae) was undertaken to increase the number of genetic markers available to breeders and to students of Lotus phylogeny. Twenty-one enzymes were examined using starch gel electrophoresis and nine buffer systems. Clear, consistent banding patterns were obtained for PGI, TPI, MDH, IDH (NADP), PGM, 6-PGDH, and ME. Clear but inconsistent banding patterns were obtained for FDP, G3PDH (NADP), ß-EST, LAP, MDH, DIA, and NADHDH. Phenotypes of the seven consistently resolved enzyme systems were obtained for different tissues for each of several genotypes at different stages of development. Variation in enzyme phenotypes of the same individuals under different growth conditions indicated the presence of different isozymic forms of these enzymes. Shoot tissue of plants over 6 weeks of age was found to be suitable material for further genetic studies, since phenotype for this tissue was constant despite changes in growing conditions. A formal genetic analysis of segregation and/or recombination of allozymes for the enzymes PGM, TPI, MDH, IDH, and 6-PGDH was undertaken. Isoenzyme phenotypes were examined for the diploids L. alpinus Schleich., L. burttii Sz. Borsos, L. conimbricensis Brot., L. ornithopodioides L., L. tennis Waldst. et Kit., and L. uliginosus Schkuhr; and for the diploid interspecific hybrids L. alpinus x L. conimbricensis, L. burttii x L. ornithopodioides, and L. japonicus x L. alpinus. Several new loci were identified for Lotus, namely, Idh1, Idh2, Mdh3, Pgi1, Pgi2, Tpi1, Tpi2, and 6-Pdgh1. Duplications of loci of IDH, MDH, PGI, and 6-PGDH were detected in the diploid (2n=12) interspecific hybrid L. japonicus x L. alpinus.

Evaluation of hypotheses concerning the origin of Lotus corniculatus (Fabaceae) using isoenzyme data.

An isoenzyme survey was conducted for several geographically dispersed accessions of four diploid Lotus species, L. alpinus Schleich., L. japonicus (Regel) Larsen, L. tenuis Waldst. et Kit and L. uliginosus Schkuhr, and for the tetraploid L. corniculatus L., in order to ascertain whether isoenzyme data could offer additional evidence concerning the origin of L. corniculatus. Seven enzyme systems were examined using horizontal starch gel electrophoresis. These were PGI, TPI, MDH, IDH, PGM, 6-PGDH, and ME. Lotus uliginosus had monomorphic unique alleles, that were not found within L. corniculatus, at 7 loci. These loci and alleles are: Tpi1-112, Pgm1,2-110, Pgm3-82, Mdh3-68, 6-Pgdh1-110, 6-Pgdh2-98,95, and Me2-100. Other diploid taxa contained alleles found in L. corniculatus for these and other loci. The implications of the isoenzyme data to theories on the origin of L. corniculatus are discussed.

Clastogenic and physiological response of chromosomes to nine pesticides in the Vicia faba in vivo root tip assay system.

9 common pesticides were assayed for clastogenic and physiological activity using Vicia faba as a eukaryotic, whole-organism, test system. The compounds tested included the insecticides acephate, demeton, monocrotophos, parathion-methyl, and trichlorfon; the fungicides captan and folpet; and the herbicides bromacil and simazine. The chemicals have been grouped according to relative genotoxicity (strongly positive: demeton, parathion-methyl; positive: folpet, acephate, monocrotophos, captan; weakly positive: bromacil, trichlorfon, simazine). The results were compared with those reported from other assay systems.

Chromosome aberration assays in Allium. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for the clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction (i.e., causing chromosome aberrations), 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.