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.

Human hypoxanthine-guanine phosphoribosyltransferase. Demonstration of structural variants in lymphoblastoid cells derived from patients with a deficiency of the enzyme.

We have explored the possibility of using cultured lymphoblasts from patients with a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a source of cells for the isolation and characterization of mutant forms of the enzyme. HPRT from lymphoblasts derived from six male patients of five unrelated HPRT-deficient families was highly purified and characterized with regard to: (a) level of immunoreactive protein, (b) absolute specific activity, (c) isoelectric point, (d) migration during nondenaturing polyacrylamide gel electrophoresis, and (e) apparent subunit molecular weight. There experiments were performed on small quantities of lymphoblasts using several micromethods involving protein blot analysis of crude extracts as well as isolation and characterization of enzyme labeled in culture with radioactive amino acids. The lymphoblast enzymes from four of the patients exhibited structural and functional abnormalities that were similar to the recently described abnormalities found with the highly purified erythrocyte enzymes from these same patients. In addition, a previously undescribed HPRT variant was isolated and characterized from lymphoblasts derived from two male siblings. This unique variant has been called HPRT Ann Arbor. We conclude that lymphoblastoid cell lines can be used as a source of cells for the detection, isolation, and characterization of structural variants of human HPRT.

Hypoxanthine:guanine phosphoribosyltransferase activity in xenografts of human osteosarcoma.

It has recently been reported that human osteosarcomas may lack the purine salvage pathway enzyme, hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8). We have established a quantitative assay for measurement of this enzyme in human osteosarcoma xenografts with analysis of products by thin-layer chromatography. Nucleotidase or phosphatase activity was readily detected and could be abolished by preheating cytosol at 60 degrees C for 10 min and performing the assay at pH 10. Alternatively, the use of 25 mM NaF at pH 7.4 also inhibited this activity. The pH optimum for this enzyme in red blood cell sonicates and tumor cytosols was pH 10. All six human osteosarcoma xenografts contained hypoxanthine:guanine phosphoribosyltransferase activity ranging from 0.97 to 4.06 nmol/min/mg of protein at pH 7.4. Control human red blood cell sonicates demonstrated activity of 0.83 nmol/min/mg of protein. These data demonstrate that human osteosarcoma xenografts contain substantial activities of this purine salvage pathway enzyme.

Decreased concentration of xanthine dehydrogenase (EC 1.1.1.204) in rat hepatomas.

Xanthine dehydrogenase (EC 1.1.1.204), the rate-limiting enzyme of purine degradation, was purified 642-fold to homogeneity from liver of male Wistar rats. Antibody was generated to the purified enzyme in white rabbits and was partially purified. For the immunotitration a radioassay of high sensitivity was developed to determine low enzyme activities. Titration curves with the antibody showed that the xanthine dehydrogenase enzyme protein amounts in slowly growing hepatoma 20 and rapidly growing hepatoma 3924A were 34 and 4% of those of normal liver, which was in good agreement with the decrease in the activity of the enzyme to 33 and 2%, respectively. The contents of flavin adenine dinucleotide, the essential cofactor of the enzyme, in the immunoprecipitates in hepatomas 20 and 3924A were 27 and 4% of that of the normal liver. This is the first report to provide immunological evidence that a decreased enzyme activity in rat hepatomas, that of xanthine dehydrogenase, was due to a decrease in the enzyme protein amount. The markedly decreased xanthine dehydrogenase activity and amount have far-reaching biochemical and pharmacological implications for the tumors.

Biochemical differences among four inosinate dehydrogenase inhibitors, mycophenolic acid, ribavirin, tiazofurin, and selenazofurin, studied in mouse lymphoma cell culture.

The mechanism of the cellular toxicity of four inosinate dehydrogenase (IMP-DH) inhibitors with different antitumor and antiviral pharmacological profiles was investigated in mouse lymphoma (S-49) cell culture. Drug effects on cell growth, nucleotide pools, and DNA and RNA synthesis were measured in the presence and absence of guanine salvage supplies. Both guanine and guanosine were capable of bypassing the IMP-DH block, while they also demonstrated some growth-inhibitory effects when added alone in high concentrations. All four drugs reduced cellular guanosine triphosphate levels and caused secondary changes of the uridine, cytidine, and adenosine triphosphate pools that were similar among the four drugs. However, several drug effects in addition to IMP-DH inhibition were observed except with mycophenolic acid which may represent a pure IMP-DH inhibitor. Both tiazofurin and selenazofurin interfered with the uptake and/or metabolism of uridine and thymidine tracers; however, this effect appeared not to contribute to their cellular toxicity in vitro. Moreover, selenazofurin and tiazofurin impaired the utilization of exogenous guanine salvage supplies for DNA and RNA synthesis, and guanine was particularly ineffective in reversing the toxic effects of tiazofurin on cell growth. This finding is important in view of the available guanine salvage supplies in vivo. Since tiazofurin, selenazofurin, and their known metabolites failed to inhibit hypoxanthine-guanine-phosphoribosyl transferase, guanosine monophosphate kinase, and guanosine diphosphate kinase in cell extracts or permeabilized cells, these drugs may interfere with salvage transport across cellular membranes. The toxic effects of mycophenolic acid and ribavirin were similarly reversed by salvage supplies of up to 200 microM guanine, which suggests that ribavirin primarily acts as an IMP-DH inhibitor under these conditions. This result could explain the rather low antitumor efficacy of both mycophenolic acid and ribavirin in vivo. However, increasing the guanine salvage supply in the medium above 200 microM further reversed the toxic effects of mycophenolic acid to maximum rescue, while it increased the toxicity of ribavirin (300 microM). This finding suggests the presence of a toxic mechanism of ribavirin at higher concentrations that is dependent upon the presence of guanine supplies sufficient to fully overcome the IMP-DH inhibition. This study documents that each antimetabolite displays a unique spectrum of activities with multiple toxic targets.

Purine nucleotide metabolism: specific aspects in chronic lymphocytic leukemia lymphocytes.

The metabolism of purine nucleotides was studied in human peripheral blood lymphocytes from healthy subjects and patients with B-cell chronic lymphocytic leukemia. Nucleotide content was determined by HPLC. The rate of de novo synthesis of purine nucleotides was measured kinetically by following the incorporation of 14C-formate into the nucleotides of a lymphocyte suspension. The patterns of the main enzymes involved in purine nucleotide metabolism (those of the salvage pathway and catabolism) were estimated by a radiochemical method. Although the data expressed in relation to cells and protein showed some discrepancies, several common differences were evident in both cases. The main differences were an increase in NAD and IMP, a sharp decrease in 5'-nucleotidase activities and in total guanylate content and synthesis, and an increase in the A/G ratio in lymphocytes of patients with respect to controls. The changes in these parameters in CLL indicate an imbalance in purine metabolism and may play a specific role in the biology of the leukemia cell. They are also potential biochemical markers of lymphoid malignancies and may be useful in chemotherapic applications.

Selection of reference genes for gene expression studies in human neutrophils by real-time PCR.

BACKGROUND: Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances. Thus the selection of reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils. RESULTS: Initial screening of the expression pattern demonstrated that 3 of the 10 genes were expressed at very low levels in neutrophils and were excluded from further analysis. The range of expression stability of the other 7 genes was (from most stable to least stable): GNB2L1 (Guanine nucleotide binding protein, beta polypeptide 2-like 1), HPRT1 (Hypoxanthine phosphoribosyl transferase 1), RPL32 (ribosomal protein L32), ACTB (beta-actin), B2M (beta-2-microglobulin), GAPD (glyceraldehyde-3-phosphate dehydrogenase) and TBP (TATA-binding protein). Relative expression levels of the genes (from high to low) were: B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1. CONCLUSION: Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.

Cell imaging and morphology: application to studies of inherited purine metabolic disorders.

A number of inherited or drug-induced metabolic disorders involving dysfunctions in purines and pyrimidines are strongly associated with neurological dysfunction, e.g., Lesch Nyhan syndrome. Such disorders have been studied extensively using biochemical and molecular techniques in order to examine how such defects occur, sometimes using in vitro models based upon cultured neuroblastoma cell lines. However, these metabolic dysfunctions may manifest their effects in other ways, such as impaired synaptic transmission and gross abnormalities in neuronal growth and differentiation. This review outlines the latter novel facet of purine research. It is proposed that by employing cell imaging techniques and cultured neuroblastoma cell lines, believed to model the nervous system, significant insights into how inherited disorders of purine metabolism affect neuronal development can be obtained. This review provides an example of the application of these techniques to understand the etiology of Lesch Nyhan syndrome, and encourages further study of the role of purines and pyrimidines in the development of the nervous system.

Changes in permissiveness for the expression of microinjected DNA during the first cleavages of mouse embryos.

LacZ DNA and LacZ RNA were microinjected during the first cleavages of embryos. LacZ DNA was not expressed before 18-19 h post insemination (hpi) but LacZ RNA was translated. Before 22 hpi LacZ DNA was expressed in the pronuclei of the one-cell embryos and the polypeptides of the minor, but not the major activation period of the genome were synthesized. This suggests a negative control of transcription before 18-19 hpi and demonstrates that its resumption is independent of the first cleavage and of the major activation of the genome. At the time of the minor activation the eggs contain the trans-acting elements to express a variety of genes that they do not express. It may indicate that, the minor and the major activation of the genome are differently controlled.

Purification and characterization of hypoxanthine/guanine phosphoribosyltransferase in bovine snout epidermis.

Hypoxanthine/guanine phosphoribosyltransferase was purified from bovine snout epidermis, about 600-fold by a combination method of centrifugation, ammonium sulfate fraction, Sephadex G-200 and DEAE cellulose chromatography. Enzymatic properties of the purified enzyme were determined as follows: pH optimum 7.2, temperature optimum 56 degrees C, and 82,000 in molecular weight. In the presence of phosphoribosyl pyrophosphate, the enzyme was extremely heat-stable. The enzyme displayed Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosyl pyrophosphate of 1.59, 20.4 and 72.6 microM respectively.

Purification of hypoxanthine-guanine phosphoribosyltransferase of Plasmodium lophurae.

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was isolated from the malarial parasite, Plasmodium lophurae. The apparent pI, as determined by chromatofocusing, was 7.6. The native molecular weight was 79,000. The pH profile of HGPRT exhibited a broad pH optimum. With hypoxanthine as substrate maximal activity was achieved from pH 6.0-10.0, and with guanine as substrate maximal activity occurred from pH 7.5-9.5. The enzyme exhibited Michaelis-Menten kinetics with all substrates. The Km values were 3.8 microM (hypoxanthine), 2.4 microM (guanine), 6.2 microM (6-mercaptopurine), 7.6 microM (6-thioguanine), and 360 microM (8-azahypoxanthine). 6-Thioinosine, 9-beta-arabinofuranosylhypoxanthine, 6-chloropurine, xanthine and azaguanine were inhibitors of the P. lophurae enzyme. From the substrate and inhibitor data it appears that the sixth position on the purine ring plays a major role in enzyme activity.

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius.

The activity of three enzymes involved in the salvage pathway of purine nucleosides--purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)--was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream.

Methapyrilene is inactive in the hepatocyte-mediated Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase mutational assay.

Methapyrilene, tested at both non-hepatocytotoxic and hepatocytotoxic concentrations, failed to induce somatic mutations in the hepatocyte-mediated Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutational assay. These data support the conclusion that methapyrilene induces the carcinogenesis process through a non-DNA damaging mechanism, suggested when this antihistamine previously failed to induce a genotoxic response in several in vitro test systems developed to detect carcinogens and mutagens.

Nitrate contamination of drinking water: relationship with HPRT variant frequency in lymphocyte DNA and urinary excretion of N-nitrosamines.

We studied peripheral lymphocyte HPRT variant frequency and endogenous nitrosation in human populations exposed to various nitrate levels in their drinking water. Four test populations of women volunteers were compared. Low and medium tap water nitrate exposure groups (14 and 21 subjects) were using public water supplies with nitrate levels of 0.02 and 17.5 mg/l, respectively. Medium and high well water nitrate exposure groups (6 and 9 subjects) were using private water wells with mean nitrate levels of 25 and 135 mg/l, respectively. Higher nitrate intake by drinking water consumption resulted in a dose-dependent increase in 24-hr urinary nitrate excretion and in increased salivary nitrate and nitrite levels. The mean log variant frequency of peripheral lymphocytes was significantly higher in the medium well water exposure group than in the low and medium tap water exposure groups. An inverse correlation between peripheral lymphocyte labeling index and nitrate concentration of drinking water was observed. Analysis of N-nitrosamine in the urine of 22 subjects by gas chromatography-mass spectrometry revealed the presence of N-nitrosopyrrolidine in 18 subjects. Analysis of the mutagenicity of well water samples showed that a small number of the well water samples were mutagenic in the Ames Salmonella typhimurium test after concentration over XAD-2 resin. In conclusion, consumption of drinking water, especially well water, with high nitrate levels can imply a genotoxic risk for humans as indicated by increased HPRT variant frequencies and by endogenous formation of carcinogenic N-nitroso compounds from nitrate-derived nitrite.

Molecular analyses of in vivo hprt mutations in human T-lymphocytes: IV. Studies in newborns.

In order to characterize in vivo gene mutations that occur during fetal development, molecular analyses were undertaken of mutant 6-thioguanine resistant T-lymphocytes isolated from placental cord blood samples of 13 normal male newborns. These mutant T-cells were studied to define hypoxanthine-guanine phosphoribosyltransferase (hprt) gene structural alterations and to determine T-cell receptor (TCR) gene rearrangement patterns. Structural hprt alterations, as shown by Southern blot analyses, occurred in 85% of these mutant clones. These alterations consisted mostly of deletion of exons 2 and 3. These findings contrast with the 10-20% of gross structural alterations (i.e., those visible on Southern blots) occurring randomly across the entire gene previously reported for T-cell mutants isolated from normal young adults. Iterative analyses of hprt structural alterations and TCR gene rearrangement patterns show that approximately one-third of the newborn derived mutants may have originated as pre- or intrathymic hprt mutations. This too contrasts with previous findings in adults where the background in vivo hprt mutations appeared to originate in postthymic T-lymphocytes.

Mice deficient for 5-lipoxygenase, but not leukocyte-type 12-lipoxygenase, display altered immune responses during infection with Schistosoma mansoni.

Periovular granuloma formation during Schistosoma mansoni infection is a complex, multifaceted immunologic response. Products of arachidonic acid metabolism have been shown to contribute to this response through studies in which general inhibitors of lipoxygenase function reduce granulomatous inflammation. To determine which lipoxygenases are important for granuloma development in schistosomiasis, wild type mice or mice deficient for 5-lipoxygenase (5-LO) or "leukocyte-type" 12-lipoxygenase (12-LO) were infected with S. mansoni and studied for responses to schistosome eggs and egg antigens. At the acute stage of infection, when granuloma formation is usually maximal, 5-LO deficient mice developed smaller granulomas around liver-deposited schistosome eggs compared with wild type or 12-LO deficient mice. 5-LO mice also displayed less antibody-mediated (5 h) and cell-mediated, delayed-type (24 h) hypersensitivity to schistosome egg antigens than did the other two infection groups. In an attempt to determine possible mechanisms for the reduced inflammatory responses, we also measured hepatic mRNA levels of cytokines that have been shown to influence granuloma size (IL-4, IL-10, and IFN-gamma). The mRNA levels for IL-10 were significantly lower in 5-LO-deficient mice, but SEA-stimulated spleen cells did not demonstrate a significant difference in IL-10 production between wild type and 5-LO mice. These data suggest that 5-LO plays a role in host responses to schistosomiasis via a mechanism that cannot be explained solely by changes in expression of these cytokines.

Reverse effect of guanine on the inhibitory action of mycophenolic acid during nucleic acid synthesis.

Mycophenolic acid (MPA) was demonstrated to inhibit DNA and RNA synthesis in L1210 cells strongly; however these effects were remarkably reduced by guanine. The presence of MPA in the medium decreased the guanine nucleotide contents (GMP, GDP, GTP) of the cells, but the addition of guanine reversed this effect. We have reported previously that MPA had no inhibitory effect on hypoxanthine guanine phosphoribosyltransferase (HGPRTase) activity. Together these findings suggest that the decrease of guanine nucleotides induced by MPA is restored by GMP, which is formed from guanine by HGPRTase in the cells. It is speculated that a suppressor of HGPRTase activity, such as 6-mercaptopurine, may protect the antitumor activity of MPA by preventing the conversion of guanine to GMP.

A simple autoradiographic method for checking HGPRT-deficiency in colonies of mammalian cells.

HGPRT enzyme activity in mutant colonies selected in 6-thioguanine can be assessed directly in petri dishes using an autoradiographic method. The application of this method to large-scale quantitative mutation experiments verifies that: (a) the maximum induced frequency of mutation of HGPRT-deficiency can be measured at short expression times, such as 3 days after treatment, when cells are respread into relatively low thioguanine concentrations, and (b) ionizing radiation induces predominantly mutants with zero HGPRT activity. Other potential applications of this method are discussed.

Some aspects of purine nucleotide metabolism in lymphocytes of B-CLL.

The authors studied the behavior of some enzymes involved in purine nucleotide metabolism in human peripheral blood lymphocytes from normal and B-cell chronic lymphocytic leukemia subjects. Determinations were made with radiochemical methods associated with high performance liquid chromatography. Results indicated a marked increase in de novo purine synthesis enzymes, particularly those of the "inosinic branch point". The latter were absent in normal lymphocytes, whereas they were well evident in leukemic lymphocytes, with the exception of AMP-S synthetase. Whereas the enzymes of the "salvage pathway" were spared in comparison to other proteins, those of the "catabolic pathway" significantly decreased. The authors discuss the possibility that such enzymes may be used as tumor markers.

[The genetic diagnosis of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency. A study of 12 cases]. / Diagnóstico genético de la deficiencia de hipoxantina-guanina fosforribosiltransferasa (HGPRT). Estudio de 12 casos.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency is transmitted as an X-linked recessive trait. Female carriers are asymptomatic and the carrier diagnosis is usually performed by determining HGPRT activity in hair roots. This technique does not allow a non-carrier state diagnosis with absolute certainty and has other limitations such as obtaining non-viable hair roots. The knowledge of the genetic mutation in three Spanish families with HGPRT deficiency, enabled us to perform the genetic diagnosis of the carrier state in 10 female subjects at risk and one female fetus. The genetic diagnosis has been performed by analyzing the differences between the mutant and the normal allele with respect to the restriction pattern. When the restriction pattern showed no differences, this has been created by directed mutagenesis. With this methodology we confirmed that a newborn of a known carrier female of HGPRT deficiency was healthy. In all cases the diagnosis could be established with great fiability in a mean time of 24 to 48 hours. We report the first genetic diagnosis of the carrier state for the HGPRT deficiency performed in Spain.