Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4 h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4 h of exposure. Whole transcriptomes were profiled after 4 h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.

Exposure to environmentally-relevant concentrations of hexavalent chromium does not induce ovarian toxicity in mice.

Exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water (≥250 ppm) is reported to decrease ovarian follicle counts and increase follicular atresia in mice. To assess effects at lower concentrations, herein we exposed B6C3F1 mice to 0.1-150 ppm Cr(VI) in drinking water for 90 days in a GLP-compliant study. Ovarian follicular counts, differentiation, and degeneration were assessed from every 10th serial section (up to 14 sections per ovary). Ovarian follicular counts, differentiation, and rate of atresia were not altered in any exposure group. Gross and microscopic changes were not apparent in any of the evaluated reproductive or glandular organs. The no observable adverse effect level (NOAEL) for follicular effects was 150 ppm. In addition to these findings, published Cr(VI) studies examining follicles were scored using two methods for assessing study quality for use in risk assessment-including the Toxic Substance Control Act (TSCA) scoring method. Both methods revealed that studies reporting adverse effects on follicles generally received low scores. Overall, the current study indicates no/low potential for Cr(VI) to induce follicular toxicity in mice below 150 ppm Cr(VI) in drinking water (17.7 mg/kg bodyweight).

Transcriptional profiling of male CD-1 mouse lungs and Harderian glands supports the involvement of calcium signaling in acrylamide-induced tumors.

Acrylamide (AA) exposure causes increased incidence of forestomach, lung, and Harderian gland tumors in male mice. One hypothesized mode of action (MOA) for AA-carcinogenicity includes genotoxicity/mutagenicity as a key event, possibly resulting from AA metabolism to the direct genotoxic metabolite glycidamide. Alternatively, altered calcium signaling (CS) has been proposed as a central key event in the MOA. To examine the plausibility of these proposed MOAs, RNA-sequencing was performed on tumor target tissues: Harderian glands (the most sensitive tumor target tissue in the rodent 2-year cancer bioassay) and lungs of AA-exposed male CD-1 mice. Animals were exposed to 0.0, 1.5, 3.0, 6.0, 12.0, or 24.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. We observed a pronounced effect on genes involved in CS and cytoskeletal processes in both tissues, but no evidence supporting a genotoxic MOA. Benchmark dose modeling suggests transcriptional points of departure (PODs) of 0.54 and 2.21 mg/kg bw-day for the Harderian glands and lungs, respectively. These are concordant with PODs of 0.17 and 1.27 mg/kg bw-day derived from the cancer bioassay data for these tissues in male mice, respectively. Overall, this study supports the involvement of CS in AA-induced mouse carcinogenicity, which is consistent with a recently proposed CS-based MOA in rat thyroid, and with other published reports of aberrant CS in malignant tumors in rodents and humans.

Role of pathology peer review in interpretation of the comet assay.

When a comet assay, an increasingly popular in vivo genotoxicity test, shows a positive test result, interpretation of that response requires ruling out any confounding tissue site toxicity. Since the comet assay typically uses only two or three daily doses of test agent, precursor tissue changes indicative of toxicity may be easily overlooked. Using case examples for two flavoring agents, perillaldehyde and 4,5-epoxydec-2(trans)-enal, we highlight the role of pathology peer review in verifying precursor tissue changes indicative of tissue site toxicity, thereby increasing confidence in final interpretation of comet assay results. Given global deliberation regarding safety assessment of compounds entering the marketplace, we recommend consideration of pathology peer review for equivocal and positive comet assays so that interpretations are universally consistent.

Differential genotoxicity of acrylamide in the micronucleus and Pig-a gene mutation assays in F344 rats and B6C3F1 mice.

Acrylamide is used in many industrial processes and is present in a variety of fried and baked foods. In rodent carcinogenicity assays, acrylamide exposure leads to tumour formation at doses lower than those demonstrated to induce genotoxic damage. We evaluated the potential of acrylamide to induce structural DNA damage and gene mutations in rodents using highly sensitive flow cytometric analysis of micronucleus and Pig-a mutant frequencies, respectively. Male F344 rats and B6C3F1 mice were administered acrylamide in drinking water for 30 days at doses spanning and exceeding the range of acrylamide exposure tested in cancer bioassays-top dose of 12.0 and 24.0mg/kg/day in mice and in rats, respectively. A positive control, N-ethyl-N-nitrosourea, was administered at the beginning and end of the study to meet the expression time for the two DNA damage phenotypes. The results of the micronucleus and Pig-a assays were negative and equivocal, respectively, for male rats exposed to acrylamide at the concentrations tested. In contrast, acrylamide induced a dose-dependent increase in micronucleus formation but tested negative in the Pig-a assay in mice. Higher plasma concentrations of glycidamide in mice than rats are hypothesized to explain, at least in part, the differences in the response. Benchmark dose modelling indicates that structural DNA damage as opposed to point mutations is most relevant to the genotoxic mode of action of acrylamide-induced carcinogenicity. Moreover, the lack of genotoxicity detected at <6.0mg/kg/day is consistent with the notion that non-genotoxic mechanisms contribute to acrylamide-induced carcinogenicity in rodents.

Adopting Duplex Sequencing™ Technology for Genetic Toxicity Testing: A Proof-of-Concept Mutagenesis Experiment with N-Ethyl-N-Nitrosourea (ENU)-Exposed Rats.

Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 10 7 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently used in vivo gene mutation assays. HIGHLIGHTS: DuplexSeq is an ultra-accurate NGS technology that directly quantifies mutationsENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissuesMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposureResults obtained with DuplexSeq were highly concordant between laboratoriesThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology.

Adopting duplex sequencing technology for genetic toxicity testing: A proof-of-concept mutagenesis experiment with N-ethyl-N-nitrosourea (ENU)-exposed rats.

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. The resulting background of less than one artifactual mutation per 107 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DS-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues⁠, a considerable advancement compared to currently used in vivo gene mutation assays.

A cross-sectional clinical study in women to investigate possible genotoxicity and hematological abnormalities related to the use of black cohosh botanical dietary supplements.

Black cohosh (BC; Actaea racemosa L.), a top-selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose-related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross-sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC-naïve women. Twenty-three women were enrolled in the BC-exposed group and 28 in the BC-naïve group. Use of any brand of BC-only supplement for at least 3 months was required for inclusion in the BC-exposed group. Supplements were analyzed for chemical composition to allow cross-product comparisons. All participants were healthy, with no known exposures (e.g., x-rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.

Absence of genotoxicity of purified Aloe vera whole leaf dry juice as assessed by an in vitro mouse lymphoma tk assay and an in vivo comet assay in male F344 rats.

Hydroxyanthracene derivatives (HAD) are naturally present in the latex layer of Aloe vera leaf, predominantly as aloins A, B and aloe-emodin. HAD are typically removed from commercial ingestible aloe products through activated charcoal filtration (decolorization). Current research aimed to evaluate genotoxic potential of a purified aloe whole leaf dry juice containing 0.3 ppm of total aloins and non-detectable aloe-emodin (LOD =0.01 ppm) in the L5178Y mouse lymphoma assay (MLA; OECD 490) and in vivo comet assay (OECD 489). No marked increases in mutant frequency at the tk locus were observed in the MLA at concentrations up to 5000 µg/mL for 3 h and 24 h (-S9), and up to a precipitating concentration of 3000 µg/mL for 3 h (+S9) compared to concurrent vehicle control. Relative total growth at the highest analyzable concentrations at 3 h (±S9) and 24 h (-S9) ranged from 64 to 133 %. In the comet assay, no statistically significant increases in DNA strand breaks were detected in the colon or kidney following oral gavage of 500, 1000 or 2000 mg/kg/day in male F344 rats for 2 days compared to concurrent vehicle control. Overall, these findings demonstrated the test article containing minimal HAD is not genotoxic under the described experimental conditions.

Transcriptomic pathway and benchmark dose analysis of Bisphenol A, Bisphenol S, Bisphenol F, and 3,3',5,5'-Tetrabromobisphenol A in H9 human embryonic stem cells.

Bisphenol A (BPA) is a chemical used in the manufacturing of plastics to which human exposure is ubiquitous. Numerous studies have linked BPA exposure to many adverse health outcomes prompting the replacement of BPA with various analogues including bisphenol-F (BPF) and bisphenol S (BPS). Other bisphenols are used in various consumer applications, such as 3,3',5,5'-Tetrabromobisphenol A (TBBPA), which is used as a flame retardant. Few studies to date have examined the effects of BPA and its analogues in stem cells to explore potential developmental impacts. Here we used transcriptomics to investigate similarities and differences of BPA and three of its analogues in the estrogen receptor negative, human embryonic stem cell line H9 (WA09). H9 cells were exposed to increasing concentrations of the bisphenols and analyzed using RNA-sequencing. Our data indicate that BPA, BPF, and BPS have similar potencies in inducing transcriptional changes and perturb many of the same pathways. TBBPA, the least structurally similar bisphenol of the group, exhibited much lower potency. All bisphenols robustly impacted gene expression in these cells, albeit at concentrations well above those observed in estrogen-positive cells. Overall, we provide a foundational data set against which to explore the transcriptional similarities of other bisphenols in embryonic stem cells, which may be used to assess the suitability of chemical grouping for read-across and for preliminary potency evaluation.

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

Evaluation of the genotoxicity of cell phone radiofrequency radiation in male and female rats and mice following subchronic exposure.

The National Toxicology Program tested two common radiofrequency radiation (RFR) modulations emitted by cellular telephones in a 2-year rodent cancer bioassay that included interim assessments of additional animals for genotoxicity endpoints. Male and female Hsd:Sprague Dawley SD rats and B6C3F1/N mice were exposed from Gestation day 5 or Postnatal day 35, respectively, to code division multiple access (CDMA) or global system for mobile modulations over 18 hr/day, at 10-min intervals, in reverberation chambers at specific absorption rates of 1.5, 3, or 6 W/kg (rats, 900 MHz) or 2.5, 5, or 10 W/kg (mice, 1,900 MHz). After 19 (rats) or 14 (mice) weeks of exposure, animals were examined for evidence of RFR-associated genotoxicity using two different measures. Using the alkaline (pH > 13) comet assay, DNA damage was assessed in cells from three brain regions, liver cells, and peripheral blood leukocytes; using the micronucleus assay, chromosomal damage was assessed in immature and mature peripheral blood erythrocytes. Results of the comet assay showed significant increases in DNA damage in the frontal cortex of male mice (both modulations), leukocytes of female mice (CDMA only), and hippocampus of male rats (CDMA only). Increases in DNA damage judged to be equivocal were observed in several other tissues of rats and mice. No significant increases in micronucleated red blood cells were observed in rats or mice. In conclusion, these results suggest that exposure to RFR is associated with an increase in DNA damage. Environ. Mol. Mutagen. 61:276-290, 2020. © 2019 Wiley Periodicals, Inc.

A profile of physiotherapy clinical education.

The purpose of the study was to examine clinical education placement data to generate a profile of providers and examine the students' exposure to health care and educational factors during clinical education. A retrospective audit of clinical placement rosters was undertaken for 3 calendar years (2001-2003). Data were analysed overall and by clinical school for sites and placements, public or private sector and type of placement. Over the 3-year period, 209 sites provided 3475 clinical placements, with the number of placements increasing from 1066 placements in 2001 to 1133 in 2002 and to 1276 in 2003. Overall, 72.2% of placements were located in metropolitan Sydney. The proportion from regional providers increased over the 3 years from 11.8% to 15.1%. Overall 85.8% of placements were delivered by public providers. The profile indicated that a considerable number of clinical sites were utilised with an emphasis on large public hospitals. The challenge for curriculum development is to reduce the clinical education demands on current providers while ensuring graduates meet entry-level standards of physiotherapy.

Comprehensive evaluation of the flavonol anti-oxidants, alpha-glycosyl isoquercitrin and isoquercitrin, for genotoxic potential.

Quercetin and its glycosides possess potential benefits to human health. Several flavonols are available to consumers as dietary supplements, promoted as anti-oxidants; however, incorporation of natural quercetin glycosides into food and beverage products has been limited by poor miscibility in water. Enzymatic conjugation of multiple glucose moieties to isoquercitrin to produce alpha-glycosyl isoquercitrin (AGIQ) enhances solubility and bioavailability. AGIQ is used in Japan as a food additive and has been granted generally recognized as safe (GRAS) status. However, although substantial genotoxicity data exist for quercetin, there is very little available data for AGIQ and isoquercitrin. To support expanded global marketing of food products containing AGIQ, comprehensive testing of genotoxic potential of AGIQ and isoquercitrin was conducted according to current regulatory test guidelines. Both chemicals tested positive in bacterial reverse mutation assays, and exposure to isoquercitrin resulted in chromosomal aberrations in CHO-WBL cells. All other in vitro mammalian micronucleus and chromosomal aberration assays, micronucleus and comet assays in male and female B6C3F1 mice and Sprague Dawley rats, and Muta™ Mouse mutation assays evaluating multiple potential target tissues, were negative for both chemicals. These results supplement existing toxicity data to further support the safe use of AGIQ in food and beverage products.

Genotoxicity evaluation of the naturally-derived food colorant, gardenia blue, and its precursor, genipin.

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans.

Magnesium stearate, a widely-used food additive, exhibits a lack of in vitro and in vivo genotoxic potential.

Magnesium stearate is widely used in the production of dietary supplement and pharmaceutical tablets, capsules and powders as well as many food products, including a variety of confectionery, spices and baking ingredients. Although considered to have a safe toxicity profile, there is no available information regarding its potential to induce genetic toxicity. To aid safety assessment efforts, magnesium sulfate was evaluated in a battery of tests including a bacterial reverse mutation assay, an in vitro chromosome aberration assay, and an in vivo erythrocyte micronucleus assay. Magnesium stearate did not produce a positive response in any of the five bacterial strains tested, in the absence or presence of metabolic activation. Similarly, exposure to magnesium stearate did not lead to chromosomal aberrations in CHL/IU Chinese hamster lung fibroblasts, with or without metabolic activation, or induce micronuclei in the bone marrow of male CD-1 mice. These studies have been used by the Japanese government and the Joint FAO/WHO Expert Committee on Food Additives in their respective safety assessments of magnesium stearate. These data indicate a lack of genotoxic risk posed by magnesium stearate consumed at current estimated dietary exposures. However, health effects of cumulative exposure to magnesium via multiple sources present in food additives may be of concern and warrant further evaluation.

Transcriptional profiling of male F344 rats suggests the involvement of calcium signaling in the mode of action of acrylamide-induced thyroid cancer.

Acrylamide (AA) exposure in 2-year cancer bioassays leads to thyroid, but not liver, adenomas and adenocarcinomas in rats. Hypothesized modes of action (MOAs) include genotoxicity/mutagenicity, or thyroid hormone dysregulation. To examine the plausibility of these two or any alternative MOAs, RNA-sequencing was performed on the thyroids and livers of AA-exposed rats, in parallel with measurement of genotoxicity (blood micronucleus and Pig-a mutant frequency) and serum thyroid hormone levels, following the exposure of male Fischer 344/DuCrl rats to 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw-day in drinking water for 5, 15, or 31 days. Differentially expressed genes in both tissues provided marginal support for hormonal and genotoxic MOAs, which was consistent with negative/equivocal genotoxicity assay and marginal changes in thyroid hormone levels. Instead, there was a pronounced effect on calcium signaling/cytoskeletal genes in the thyroid. Benchmark dose modeling of RNA-sequencing data for the calcium signaling pathway suggests a point of departure (POD) of 0.68 mg/kg bw-day, which is consistent with a POD of 0.82 mg/kg bw-day derived from the thyroid 2-year cancer bioassay data. Overall, this study suggests a novel MOA for AA-induced thyroid carcinogenicity in male rats centered around perturbation of calcium signaling.

Physiotherapy students' attitudes towards and knowledge of older people.

The purpose of this study was to determine whether students' attitudes towards and knowledge of older people changed throughout the physiotherapy undergraduate program. Students' demographic information and attitudes towards and knowledge of older people were gathered via repeated question-responses over three points of time during the 4-year program. Validated instruments--the Geriatric Attitudinal Scale (GAS) and the Facts on Ageing Quiz 1 (FAQ1)--were used to measure participants' attitudes and knowledge of older people. The FAQ1 yielded two variables: knowledge as percentage of FAQ1 correct responses, and ignorance as percentage of FAQ1 Don't Know responses. The instruments were administered in Year 2 prior to the students' first clinical (Time 1), immediately after their first clinical (Time 2), and Year 4 just prior to completion of the program (Time 3). Changes over time were analysed using paired t-tests with significance set at p < 0.05. Participants initially demonstrated positive attitudes towards older people with a mean of 73.8% GAS positive responses with no significant change over time (p = 0.56). Initial knowledge about older people was poor with a mean of 43.6% FAQ1 correct responses which increased significantly over time to 51.7% (p = 0.0001). This improvement in knowledge was accompanied by a decrease in ignorance over the study period (p = 0.0001). While attitudes toward older people were positive and knowledge improved over time, the level of knowledge attained was below expectation for beginning practitioners. This finding has implications for the education and training of physiotherapy students.

Deregulation of polyamine biosynthesis alters intrinsic histone acetyltransferase and deacetylase activities in murine skin and tumors.

The essential requirement for polyamines for normal cell growth and differentiation may be partly attributed to their influence on gene expression, a process regulated by the acetylation state of nucleosomal histones. We used transgenic mice to examine the effects of constitutive expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on histone acetylation in epithelial cells in skin. As compared with the skin of normal littermate mice, both intrinsic histone acetyltransferase (HAT) and deacetylase activities are elevated in ODC transgenic skin. Skin tumors that form spontaneously in ODC/Ras double transgenic mice exhibit exceptionally high HAT activity having a distinct specificity for Lys-12 in the tail domain of histone H4, which may have implications for gene transcription. However, acetylation of histones by HAT enzymes was impeded in cultured ODC transgenic keratinocytes, and there were only modest changes in levels of acetylated histones in the skin of ODC transgenic mice. Treatment with the ODC enzyme inhibitor alpha-difluoromethylornithine, which results in regression of ODC/Ras tumors, reverses the effects on HAT and deacetylase enzyme function, implicating polyamine biosynthesis in the regulation of histone acetylation. Polyamines do not directly stimulate the enzymatic activity of either p300 or p300/CREB-binding protein (CBP)-associated factor, members of two distinct classes of HAT enzymes, implying that the elevated CBP/p300-associated HAT activity detected in ODC transgenic skin is attributable to indirect influence of polyamines. These results suggest that multiple mechanisms exist by which endogenous polyamines influence chromatin in mammals. Furthermore, they suggest that the elevated polyamine levels inherent in many solid tumors alter chromatin structure, likely affecting gene expression and promoting the neoplastic process.

Genetic and rat toxicity studies of cyclodextrin glucanotransferase.

INTRODUCTION: Microbiologically derived cyclodextrin glucanotransferase (CGTase) is used commercially as a processing agent in manufacture of food, pharmaceuticals, and cosmetics. Its toxic potential was evaluated in anticipation of use in the production of alpha-glycosyl isoquercitrin, a water-soluble form of quercetin. METHODS: Following OECD guidelines, CGTase, produced by Bacillus pseudalcaliphilus DK-1139, was evaluated in a genotoxicity battery consisting of a bacterial reverse mutation assay, an in vitro micronucleus (MN) assay and MN and comet assays using B6C3F1 male and female mice. These same genotoxicity assays were also conducted for sodium sulfate, a contaminant of CGTase preparation. In a 90-day Sprague Dawley rat toxicity study, CGTase was administered by gavage in water at daily doses of 0, 250, 500, and 1000 mg/kg/day. RESULTS: CGTase did not induce mutations with or without metabolic activation in the bacterial reverse mutation assay. Formation of micronuclei was not induced in either in vitro or in vivo MN assays with or without metabolic activation. No induction of DNA damage was detected in male or female mouse liver, stomach, or duodenum in the comet assay. Sodium sulfate also tested negative in these same genotoxicity assays. In the 90-day repeated dose rat study there were no treatment-related adverse clinical or pathological findings. CONCLUSION: The genotoxicity assays and repeated dose toxicity study support the safe use of CGTase in production of alpha-glycosyl isoquercitrin.