HPRT Research Articles

Mutagenicity of 7-ketocholesterol in CHO cells: The role of lipid peroxidation

As an important cholesterol oxide, 7-ketocholesterol plays a deleterious role in the occurrence of cancer. Although the fact had been proved that 7-ketocholesterol could induce several biological phenomena, including apoptosis, DNA damage, et al., this issue whether 7-ketocholesterol led to mutagenesis in mammalian cells remains largely unexplored. Here, we investigated the major role of lipid peroxidation in the genotoxic response to 7-ketocholesterol in chinese hamster ovary (CHO) cells. The results showed that 7-ketocholesterol induced gene mutation and DNA double-strand breaks (DSBs) in concentration- and time-dependent manner. After CHO cells were treated with 25 μM 7-ketocholesterol for 48 h, the mutation frequency at hprt gene loci and the level of γ-H2AX protein were both significantly increased. Exposure to 7-ketocholesterol resulted in a concentration-dependent increase in the apoptotic rate and the protein expression of cleaved caspase-3 and -7 in CHO cells. Moreover, a significant increase of superoxide dismutase (SOD) activity and content of malondialdehyde (MDA) was also observed. Using a inhibitor of lipid peroxidation (butylated hydroxytoluene), it was found to remarkably inhibit the genotoxicity and MDA levels caused by 7-ketocholesterol. These findings indicated that lipid peroxidation was involved in the mutagenic process of 7-ketocholesterol in CHO cells.

Mutation in hprt1 Gene or HPRT Deficiency May be a Restricting Progeny of Favipiravir in Covid-19

Mutation in hprt1 Gene or HPRT Deficiency May be a Restricting Progeny of Favipiravir in Covid-19

Involvement of APOBEC3B in mutation induction by irradiation.

To better understand the cancer risk posed by radiation and the development of radiation therapy resistant cancer cells, we investigated the involvement of the cancer risk factor, APOBEC3B, in the generation of radiation-induced mutations. Expression of APOBEC3B in response to irradiation was determined in three human cancer cell lines by real-time quantitative PCR. Using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutation assay, mutations in the HPRT gene caused by irradiation were compared between APOBEC3B-deficient human hepatocellular carcinoma (HepG2) cells [APOBEC3B knocked out (KO) using CRISPR-Cas9 genome editing] and the parent cell line. Then, HPRT-mutated cells were individually cultured to perform PCR and DNA sequencing of HPRT exons. X-Irradiation induced APOBEC3B expression in HepG2, human cervical cancer epithelial carcinoma (HeLa) and human oral squamous cell carcinoma (SAS) cells. Forced expression of APOBEC3B increased spontaneous mutations. By contrast, APOBEC3B KO not only decreased the spontaneous mutation rate, but also strongly suppressed the increase in mutation frequency after irradiation in the parent cell line. Although forced expression of APOBEC3B in the nucleus caused DNA damage, higher levels of APOBEC3B tended to reduce APOBEC3B-induced γ-H2AX foci formation (a measure of DNA damage repair). Further, the number of γ-H2AX foci in cells stably expressing APOBEC3B was not much higher than that in controls before and after irradiation, suggesting that a DNA repair pathway may be activated. This study demonstrates that irradiation induces sustained expression of APOBEC3B in HepG2, HeLa and SAS cells, and that APOBEC3B enhances radiation-induced partial deletions.

EXPRESSION OF AQUAPORINS 1 AND 5 IN PAROTID GLANDS OF RATS AFTER VOLUMETRIC MODULATED ARC THERAPY AND LASER THERAPY

Objective: The experimental study investigated the expression of type-1 and type-5 aquaporins (AQPs) in samples from parotid glands of rats irradiated by volumetric modulated arc therapy (VMAT) and subjected to low-level laser therapy (LLLT) at different times. Study design: Thirty Wistar rats distributed in control groups without intervention (CG), only LLLT (LG) and only radiotherapy (RG), and the use of immediate LLLT 24 hours (IEG) and late 120 hours (LEG) after radiotherapy. VMAT was single dose (12 Gy), and LLLT with laser AsGaAl (660 nm, 100 mW), spot 0.0028 cm2, at 3 points in the right parotid gland region (60 seconds and 70 J/cm2/d), for 10 consecutive days until euthanasia. The parotid glands were dissected and prepared, and the gene expression of the AQPs evaluated by quantitative polymerase chain reaction (qPCR) using specific probes TaqMan and HPRT gene as constitutive controls. Results: All groups presented AQP expression without significant difference in comparison to CG; however, mean expression of AQPs 1 and 5 was higher in groups that used LLLT, even at later times. Conclusion: VMAT does not cause destruction of AQPs, and the use of late LLLT can provide maintenance and increase of these proteins that participate in the process of salivary secretion. Financial support: CAPES Objective: The experimental study investigated the expression of type-1 and type-5 aquaporins (AQPs) in samples from parotid glands of rats irradiated by volumetric modulated arc therapy (VMAT) and subjected to low-level laser therapy (LLLT) at different times. Study design: Thirty Wistar rats distributed in control groups without intervention (CG), only LLLT (LG) and only radiotherapy (RG), and the use of immediate LLLT 24 hours (IEG) and late 120 hours (LEG) after radiotherapy. VMAT was single dose (12 Gy), and LLLT with laser AsGaAl (660 nm, 100 mW), spot 0.0028 cm2, at 3 points in the right parotid gland region (60 seconds and 70 J/cm2/d), for 10 consecutive days until euthanasia. The parotid glands were dissected and prepared, and the gene expression of the AQPs evaluated by quantitative polymerase chain reaction (qPCR) using specific probes TaqMan and HPRT gene as constitutive controls. Results: All groups presented AQP expression without significant difference in comparison to CG; however, mean expression of AQPs 1 and 5 was higher in groups that used LLLT, even at later times. Conclusion: VMAT does not cause destruction of AQPs, and the use of late LLLT can provide maintenance and increase of these proteins that participate in the process of salivary secretion. Financial support: CAPES

The L.donovani Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) oligomer is distinct from the human homolog

Purine bases, synthesized de novo or recycled through the salvage pathway, are precursors of nucleotide synthesis and are essential in a variety of physiological processes including cell division, growth, signaling, energy metabolism and synthesis of vitamins/co-factor. The protozoan kinetoplastid parasites including Leishmania cannot synthesize de novo and rely solely on the purine salvage pathway, recycling the degraded products of nucleic acid metabolism. Enzymes of this pathway are thus of therapeutic importance. The enzyme Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (EC 2.4.2.8) plays a central role in this pathway, converting the purine base to its monophosphate product. Towards the elucidation of its role, we have cloned, expressed, purified and determined the crystal structure of L. donovani HGPRT at 2.76 Å. Comparative structural analysis with the human homolog indicates differences in oligomer association. Comparative analyses identify insertions in the human homolog sequence in the tetramer interface. The results suggest that this difference can be exploited for therapeutic approaches.

Differential Expression of IGF1, IGFBP5, MSTN, and MYH1 Across Different Age Classes in American Quarter Horses

The objective of this study was to determine the influence of age on expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor binding protein (IGFBP5), myostatin (MSTN), and myosin (MYH1) genes which are related to growth and muscle development in the American Quarter Horse. Thus, horses (n = 10) from weanling, yearling, 2-, 3-, and 10-year-old age classes were sampled and gene expression was assessed by RT-qPCR. ΔCT was calculated using the hypoxanthine-guanine phosphoribosyltransferase gene as an internal normalizer. The generalized linear model was used to determine differentially expressed genes, by pairwise comparison between ages. Among technical replicates, the coefficient of variation ranged from 1.0 to 5.2% and was lower than the variation observed between biological replicates (2.1–12.9%). IGF1 demonstrated significantly lower expression in the 3-year-old age class than in weanlings and yearlings, but the 10-year-old age class displayed a significantly higher level than 2- and 3-year-old age classes. Expression of IGFBP5 was highest in weanlings compared with all other age classes. Expression of MSTN was significantly higher in weanlings than in other age classes, whereas 10-year-old horses had an intermediate level of expression, but significantly different from yearlings, 2- and 3-year-old fillies. Finally, expression of MYH1 was lower in 2- and 10-year-old horses than in weanlings and yearlings, whereas 3-year-old fillies demonstrated an intermediate level of expression. Differential expression patterns observed in this preliminary study provide insight into the physiological changes occurring throughout the life span of horses. These patterns could also help explain the variation in performance and endurance between individuals at different developmental stages.

Effects of melatonin to arecoline-induced reactive oxygen species production and DNA damage in oral squamous cell carcinoma

Arecoline, the major alkaloid of areca nut, is known to induce reactive oxygen species (ROS) and DNA damage during oral cancer progression. This study aim to evaluate whether melatonin, an antioxidant, supported or repressed the arecoline-induced carcinogenesis phenotypes in oral squamous cell carcinoma (OSCC). The cytotoxicity of arecoline or melatonin treatment alone and their co-treatment in the OSCC cell line OEC-M1 were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell cycle, cell death, and total ROS production were analyzed using flow cytometer. The protein expression was determined using western blot analysis. The genotoxicity and mutation rate were determined using micronucleus assay and hypoxanthine phosphoribosyl transferase (HPRT) forward mutation assay, respectively, in CHO-K1 cells. The ataxia telangiectasia mutated (ATM) promoter activity and DNA repair ability were determined through reporter assay. The result showed that both the arecoline and melatonin induced ROS production and antioxidant enzymes expression. Melatonin treatment enhanced arecoline-induced ROS production, cytotoxicity, G2/M phase arrest, and cell apoptosis in OSCC cells. On the other hand, melatonin treatment activated DNA repair activity to reverse arecoline-induced DNA damage and mutation. These results indicated that melatonin is a potential chemopreventive agent for betel quid chewers to prevent OSCC initiation and progression.

Evaluation of the upregulation and surface expression of hypoxanthine guanine phosphoribosyltransferase in acute lymphoblastic leukemia and Burkitt\u2019s B cell lymphoma

BackgroundThe aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies. With 4.3% of all new cancers diagnosed as Non-Hodgkin lymphoma, finding new biomarkers for the treatment of B cell cancers is an ongoing pursuit. HPRT is a nucleotide salvage pathway enzyme responsible for the synthesis of guanine and inosine throughout the cell cycle.MethodsRaji cells were used for this analysis due to their high HPRT internal expression. Internal expression was evaluated utilizing western blotting and RNA sequencing. Surface localization was analyzed using flow cytometry, confocal microscopy, and membrane biotinylation. To determine the source of HPRT surface expression, a CRISPR knockdown of HPRT was generated and confirmed using western blotting. To determine clinical significance, patient blood samples were collected and analyzed for HPRT surface localization.ResultsWe found surface localization of HPRT on both Raji cancer cells and in 77% of the malignant ALL samples analyzed and observed no significant expression in healthy cells. Surface expression was confirmed in Raji cells with confocal microscopy, where a direct overlap between HPRT specific antibodies and a membrane-specific dye was observed. HPRT was also detected in biotinylated membranes of Raji cells. Upon HPRT knockdown in Raji cells, we found a significant reduction in surface expression, which shows that the HPRT found on the surface originates from the cells themselves. Finally, we found that cells that had elevated levels of HPRT had a direct correlation to XRCC2, BRCA1, PIK3CA, MSH2, MSH6, WDYHV1, AK7, and BLMH expression and an inverse correlation to PRKD2, PTGS2, TCF7L2, CDH1, IL6R, MC1R, AMPD1, TLR6, and BAK1 expression. Of the 17 genes with significant correlation, 9 are involved in cellular proliferation and DNA synthesis, regulation, and repair.ConclusionsAs a surface biomarker that is found on malignant cells and not on healthy cells, HPRT could be used as a surface antigen for targeted immunotherapy. In addition, the gene correlations show that HPRT may have an additional role in regulation of cancer proliferation that has not been previously discovered.

Biosensor-based monitoring of the central metabolic pathway metabolites

In vivo biosensors have a wide range of applications, from the detection of metabolites to the regulation of metabolic networks, providing a versatile tool for cell biology and metabolic engineering. However, compared with the vast array of small molecules present in nature, the existing range of biosensors is far from sufficient. Here we describe the use of human hypoxanthine guanine phosphoribosyltransferase (HGPRT) as a ligand binding domain (LBD) protein, that acts by ligand-dependent stabilization, to build a biosensor for detection of the pentose phosphate pathway metabolite 5-phospho-α-D-ribose 1-diphosphate (PRPP). Using this protein as a template, we computationally redesigned a new pocket de novo according to the pose of the ligand, creating a binding mode exclusive to recognize another pentose phosphate metabolite, D-erythrose 4-phosphate (E4P), and glycerate-3-phosphate (3PG), from the glycolysis pathway. Furthermore, E4P biosensor was developed by fluorescence-activated cell sorting (FACS) and application of it enabled successful screening for the highest phenylalanine-producing strain reported to date. This work provides a strategy for computational design and development of biosensors for a broad range of molecules.

Evaluation of HPRT Gene mutation and Comet Assay in Some Breast Cancer Patients Undergoing Radiotherapy

Introduction: Radiotherapy is a toxic cellular treatment that destroys rapidly dividing cells such as cancer cells. The strategy for HPRT mutation examination in human’s cells was commonly used to establish in vivo history along with mutation rates in children and adults inhabitants subjected to experienced and unfamiliar external mutagens. As just a practical approach to human biosurveillance studies, the comet assay has been introduced over the past years as biomarker to identify ionizing radiation Consequences with Patients undergoing radiotherapy in breast cancer. Materials and Method: This study was carried out on thirty Iraqi women patients with BC patients undergoing radiotherapy about 20-30 Gy locally gamma cells at Al-Amel National Hospital for cancer Management in Baghdad during time 2-13 years, non-smokers and non- alcoholic, aged (35-55 year), with stage (grade) I-III, as well as thirty apparently healthy individuals females collected randomly from population living Baghdad, Old age (35-55) Which are non-alcoholic non-smokers as group of control. Around the ongoing research comet-test and hprt mutation test could be applied to study damages in DNA for genetic two endpoints For patients with cancer throughout radiation therapy. Results: The present study showed significant increase (p<0.05) in the HPRT gene mutation assay for the in breast cancer patients undergoing radiotherapy as compared with the control group. Also there were found that the values of comet tail moment and tail length was increased significantly (p<0.05) in the human lymphocyte in these for breast cancer patients undergoing radiotherapy as compared with the control group. Conclusions: Present results revealed that there is a probability of utilizing human lymphocytes changes as useful biodosimetric markers for the detection of human exposure to ionizing radiation, the data gathered also demonstrated the utility of the HPRT gene mutation and (Alkaline) comet test as just a precise alternative biological marker for routine preventative care of cancer patients receiving radiation therapy.

The multiplex interactions and molecular mechanism on genotoxicity induced by formaldehyde and acrolein mixtures on human bronchial epithelial BEAS-2B cells

Aldehydes are common air pollutants with carcinogenicity. Genotoxicity of single aldehyde has been studied well, but the combined genotoxicity is rarely known. Here, we evaluated the combined genotoxicity of formaldehyde and acrolein on BEAS-2B cells in terms of DNA strands breakage, chromosome damage and gene mutation below subcytotoxic concentrations covering smoking-related concentrations. Meanwhile, the molecular mechanism was investigated further based on oxidative stress, DNA-protein crosslinks (DPCs), cell cycle and DNA damage-repair pathway. Co-exposure to formaldehyde and acrolein mixtures showed significantly synergistic interaction on DNA strands breakage and chromosome damage in a concentration/time-dependent manner, while antagonism was shown on the late genotoxic endpoints (e.g. cytoplasmic block micronucleus (CBMN) and HPRT gene mutation). Moreover, formaldehyde synergistically potentiated acrolein-induced S-phase arrest, inhibition of DNA repair and up-regulation of genes related to cell stress, which conversely strengtherned mixture-induced DNA/chromosome damage and finally resulted in antagonism on late genotoxic events. Additionally, formaldehyde-induced DNA damage mainly resulted from the direct covalent bonding (e.g. DPCs), while acrolein-induced DNA damage mainly generated from oxidative damage (e.g. oxidative stress), which dominated the synergistic DNA strand breakage induced by mixtures. Summarily, aldehyde mixtures (formaldehyde and acrolein) induced multiplex combined genotoxicity on BEAS-2B cells even at smoking-related concentrations, which was dependent on genotoxic endpoints and closely related to that formaldehyde potentiated acrolein-induced cell stress, S-phase arrest and inhibition of DNA repair. So prolonged exposure to aldehyde mixtures may have a more serious risk to respiratory system in animal and human than the expectation based on the toxicity of single aldehyde even at environmentally relevant concentrations.

Molecular Mechanisms Involved in Neural Substructure Development during Phosphodiesterase Inhibitor Treatment of Mesenchymal Stem Cells.

Stem cells are highly important in biology due to their unique innate ability to self-renew and differentiate into other specialised cells. In a neurological context, treating major injuries such as traumatic brain injury, spinal cord injury and stroke is a strong basis for research in this area. Mesenchymal stem cells (MSC) are a strong candidate because of their accessibility, compatibility if autologous, high yield and multipotency with a potential to generate neural cells. With the use of small-molecule chemicals, the neural induction of stem cells may occur within minutes or hours. Isobutylmethyl xanthine (IBMX) has been widely used in cocktails to induce neural differentiation. However, the key molecular mechanisms it instigates in the process are largely unknown. In this study we showed that IBMX-treated mesenchymal stem cells induced differentiation within 24 h with the unique expression of several key proteins such as Adapter protein crk, hypoxanthine-guanine phosphoribosyltransferase, DNA topoisomerase 2-beta and Cell division protein kinase 5 (CDK5), vital in linking signalling pathways. Furthermore, the increased expression of basic fibroblast growth factor in treated cells promotes phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) cascades and GTPase–Hras interactions. Bioinformatic and pathway analyses revealed upregulation in expression and an increase in the number of proteins with biological ontologies related to neural development and substructure formation. These findings enhance the understanding of the utility of IBMX in MSC neural differentiation and its involvement in neurite substructure development.

The combination of hprt and gapdh is the best compound reference genes in the fetal rat hippocampus.

Hippocampus, as an important organ of central memory storage and spatial orientation, has been studied increasingly in recent years. The expression of reference genes in the hippocampus of adult rats, which are commonly used in the quantitative real-time polymerase chain reaction (qRT-PCR), is unstable in the fetal hippocampus and may not be suitable for the fetal period. Therefore, this study intends to screen and determine the optimal compound reference genes in the fetal rat hippocampus. Based on the literature, we selected five housekeeping genes (HKGs), including glyceraldehyde 3-phosphate dehydrogenase (gapdh), actin beta (β-actin), hypoxanthine phosphoribosyltransferase (hprt), 18s ribosomal RNA (18s rRNA), and cyclophilin B (cypB). We analyzed the expression of them under physiological conditions in the fetal rat hippocampus using BestKeeper, GeNorm, and NormFinder, to select the most stable compound reference genes. Furthermore, to verify the stability of the compound reference genes, we analyzed the expression of reference genes in the fetal rat hippocampus under the pathological model of prenatal dexamethasone exposure (PDE). Finally, we evaluated the accuracy of compound reference genes through detecting the expression of fetal rat hippocampal brain-derived neurotrophic factor (BDNF) under PDE model. This study determined that the combination of gapdh and hprt was the most stable and suitable compound reference genes in the fetal rat hippocampus. There was no significant difference between male and female fetal rats. We provided the support of accurate and reliable reference genes for the further study of diseases related to the fetal hippocampus.

Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp.

Acanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism. Many open questions can be addressed by gene expression studies, however, for Acanthamoeba reliable standards have not yet been established. In this study, suitable reference genes (RGs) for RT-qPCR in Acanthamoeba were comprehensively evaluated, comparing different Acanthamoeba strains and employing four different algorithms (NormFinder, GeNorm, BestKeeper and RefFinder). Expression stability was assessed under various conditions and the potentials of the most promising RGs for accurate normalization of target genes were evaluated. Expression stability of RGs varied depending on conditions and employed algorithms, however, the genes for the 18S rRNA and the hypoxanthine phosphoribosyl transferase seem to be widely suitable RGs. Normalization with a combination of two carefully chosen RGs resulted in reliable expression data for target genes, while normalization with unsuitable RGs led to significant misinterpretation of expression profiles. Thus, a careful evaluation of RGs prior to expression studies is essential.

Hydrogen Bond Studies in Interact 6-Mercaptopurine with its Receptor Hypoxanthine-Guanine Phosphoribosyltransferase

Objective: The aim of this study was to study some properties of the pharmaceutical compound (6-mercaptopurine) a number of theoretical methods were carried out to calculate their molecular energy, the length of the bonds and angles, in addition to their chemical forms and the binding sites with the enzyme as important anti-cancer inhibitors. Methods: The intramolecular hydrogen bond, molecular structure, and vibrational frequencies of 6- Mercaptopurine have been investigated by means of density functional (DFT and AM1) methods with 6-311++G basis. Results: The nature of these interactions, known as resonance assisted hydrogen bonds, has been discussed. As a geometrical indicator of a local aromaticity, the geometry-based HOMA index has been applied. Additionally, the linear correlation coefficients between substituent constants and selected parameters in R position have been calculated. Conclusion: The results show that the hydrogen bond strength is mainly governed by the resonance variations inside the chelate ring induced by the substituent groups. The topological properties of the electron density distributions for 6- MP H...N intra molecular bridges have been analyzed .Finally, the natural population analysis methods has been used to evaluate the hydrogen bonding interactions.

Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates

In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from Zobellia galactanivorans (ZgHGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C. Thermal denaturation experiments revealed that ZgHGPRT/AMPK exhibits an apparent unfolding temperature (Tm) of 45°C and a retained activity of around 80% when incubated at 40°C for 240 min. This bifunctional enzyme shows a dependence on divalent cations, with a remarkable preference for Mg2+ and Co2+ as cofactors. More interestingly, substrate specificity studies revealed ZgHGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of the substrate. Finally, to assess the potential of ZgHGPRT/AMPK as biocatalyst for the synthesis of nucleoside-5′-mono, di- and triphosphates, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of water-miscible solvents on enzyme activity were studied.

Evaluation of Aquaporins 1 and 5 Expression in Rat Parotid Glands After Volumetric Modulated Arc Radiotherapy and Use of Low-Level Laser Therapy at Different Times.

Introduction: This experimental study investigated the mRNA expression of aquaporins (AQPs) 1 and 5 in the parotid glands of rats irradiated with volumetric modulated arc therapy (VMAT) and subjected to low-level laser therapy (LLLT) at different time points. Methods: The sample consisted of 30 Wistar rats (Rattus norvegicus) divided into the following groups: control, LLLT alone (LG), radiotherapy alone (RG), and experimental groups that received LLLT at 24 hours (early experimental group [EEG], n=12) and 120 hours (late experimental group [LEG], n=12) after radiotherapy. VMAT was delivered at a single dose (12 Gy) and LLLT was performed with an aluminium-gallium-arsenide diode laser (660 nm, 100 mW), spot area of 0.0028 cm2, energy of 2 J/cm2 applied to 3 spots in the region corresponding to the right parotid gland, for 10 consecutive days. The right parotid gland was resected and prepared for RNA extraction. The gene expression of AQPs was evaluated by quantitative polymerase chain reaction (qPCR) using specific TaqMan probes, with the HPRT gene as an internal control. Results: The lowest AQP1 gene expression was 0.83 (0.27) with the use of LLLT 24 hours after radiotherapy (EEG), and the highest was 1.56 (0.80) with the use of LLLT alone (LG). Likewise, the lowest AQP5 gene expression was found in the EEG (mean = 0.88; SD = 0.49) and the highest in the LG (mean = 1.29; SD = 0.33). Conclusion: The use of LLLT after radiotherapy may contribute to the maintenance and an increase of these proteins, even when used at a later time point after radiotherapy.

Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.

Cytotoxicity and Mutagenicity of Narrowband UVB to Mammalian Cells.

Phototherapy using narrowband ultraviolet-B (NB-UVB) has been shown to be more effective than conventional broadband UVB (BB-UVB) in treating a variety of skin diseases. To assess the difference in carcinogenic potential between NB-UVB and BB-UVB, we investigated the cytotoxicity via colony formation assay, genotoxicity via sister chromatid exchange (SCE) assay, mutagenicity via hypoxanthine phosphoribosyltransferase (HPRT) mutation assay, as well as cyclobutane pyrimidine dimer (CPD) formation and reactive oxygen species (ROS) generation in Chinese hamster ovary (CHO) and their NER mutant cells. The radiation dose required to reduce survival to 10% (D10 value) demonstrated BB-UVB was 10 times more cytotoxic than NB-UVB, and revealed that NB-UVB also induces DNA damage repaired by nucleotide excision repair. We also found that BB-UVB more efficiently induced SCEs and HPRT mutations per absorbed energy dosage (J/m2) than NB-UVB. However, SCE and HPRT mutation frequencies were observed to rise in noncytotoxic dosages of NB-UVB exposure. BB-UVB and NB-UVB both produced a significant increase in CPD formation and ROS formation (p < 0.05); however, higher dosages were required for NB-UVB. These results suggest that NB-UVB is less cytotoxic and genotoxic than BB-UVB, but can still produce genotoxic effects even at noncytotoxic doses.