Influence of body lesion severity on oxidative status and gut microbiota of weaned pigs.

Body lesions in pigs are a common welfare concern, particularly during the weaning period. These lesions can lead to pain, infection, and impaired mobility, resulting in reduced growth performance and increased mortality. Moreover, weaning stress can affect gut microbiota, immune response and increase the oxidative stress of piglets during this transition period. It has been hypothesised that social stress and body lesions could contribute to affect the gut microbiota, physiological and immune response of piglets. The study aims to evaluate the impact of the body lesions due to social stress on microbial profile, immune response, and oxidative status of weaned piglets. Lesion score (LS) on skin, tail, ear, neck, middle trunk, and hind quarters was measured 1 week (28 days of age, T1) and 7 weeks postweaning (T2) on 45 tail-docked pigs according to the method suggested from the Walfer Quality® (2009) on a scale from 0 to 2. Based on the LS, at T1, piglets were classified as High LS (n = 16), when LS was >1 in at least two of the areas considered, or Low LS (n = 29). At T2, based on the same scoring system and to the LS observed at T1, piglets were divided into four groups: High to Low LS (H-L, n = 11), High to High LS (H-H, n = 5), Low to Low LS (L-L, n = 21) and Low to High LS (L-H, n = 8). Blood and faecal samples were collected at T1 and T2. At T1, pigs with a high LS had a lower biological antioxidant potential compared with the L group (P < 0.02). At T2, the L-H group had a lower Reactive Oxygen Metabolites concentration compared with the H-H group (P = 0.03) while the L-L group had a lower concentration of Immunoglobulin A compared with H-H and L-H groups (P = 0.02 and P = 0.04, respectively). At T1, piglets with high LS had a different microbiota compared to piglets with low LS (R2 = 0.04, P < 0.01). Low LS pigs were characterised by a higher abundance of Firmicutes, Blautia, Eubacterium coprostanoligenes, Faecalibacterium, Megasphaera, Subdoligranulum (P.adj < 0.05), while pigs with high LS were characterised by higher abundance of Bacteroidota, Rikenellaceae RC9, Prevotellaceae UCG-003, uncultured-Lachnospiraceae and uncultured-Oscillospiraceae (P.adj < 0.05). At T2, the H-H group were characterised by Oscillospirales-UCG-010, H-L by Agatobachter and L-L by Alloprevotella (P.adj < 0.05). Overall, this study provides valuable insights into the relationship between body lesions, oxidative stress, and gut microbiota in weaned pigs.

Disruption of the microbiota-gut-brain axis is a defining characteristic of the α-Gal A (-/0) mouse model of Fabry disease.

Fabry disease (FD) is an X-linked metabolic disease caused by a deficiency in α-galactosidase A (α-Gal A) activity. This causes accumulation of glycosphingolipids, especially globotriaosylceramide (Gb3), in different cells and organs. Neuropathic pain and gastrointestinal (GI) symptoms, such as abdominal pain, nausea, diarrhea, constipation, and early satiety, are the most frequent symptoms reported by FD patients and severely affect their quality of life. It is generally accepted that Gb3 and lyso-Gb3 are involved in the symptoms; nevertheless, the origin of these symptoms is complex and multifactorial, and the exact mechanisms of pathogenesis are still poorly understood. Here, we used a murine model of FD, the male α-Gal A (-/0) mouse, to characterize functionality, behavior, and microbiota in an attempt to elucidate the microbiota-gut-brain axis at three different ages. We provided evidence of a diarrhea-like phenotype and visceral hypersensitivity in our FD model together with reduced locomotor activity and anxiety-like behavior. We also showed for the first time that symptomology was associated with early compositional and functional dysbiosis of the gut microbiota, paralleled by alterations in fecal short-chain fatty acid levels, which partly persisted with advancing age. Interestingly, most of the dysbiotic features suggested a disruption of gut homeostasis, possibly contributing to accelerated intestinal transit, visceral hypersensitivity, and impaired communication along the gut-brain axis.

Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair.

Use of oligodeoxynucleotides containing O6-alkylguanine for the assay of O6-alkylguanine-DNA-alkyltransferase activity.

A sensitive assay procedure was developed for the measurement of the activity of mammalian O6-alkylguanine-DNA-alkyltransferase. The procedure utilized oligodeoxynucleotides containing O6-methylguanine as substrates for the reaction. The oligodeoxynucleotides were end labeled with 32P by the reaction with polynucleotide kinase and [gamma-32P]ATP and allowed to react with organ or cell extracts containing the alkyltransferase. The unmethylated product which was formed was separated from the substrate by reverse-phase high-pressure liquid chromatography. Since the repair by the alkyltransferase is bimolecular, the second order rate constants for the reaction between the labeled oligomer and repair protein from several different sources were determined. The amount of alkyltransferase present was then calculated from the amount of product formed and the appropriate second order rate constant for the reaction. Excellent agreement was obtained between the alkyltransferase levels determined in this procedure and those measured by conventional assay procedures in a variety of cell lines having both high and low activity. The method also gave results in good agreement with other assay procedures for a number of rat tissues, although a few tissues gave anomalous results owing to a high level of nuclease activity which degraded the substrate. This method should prove useful for the measurement of alkyltransferase activity in samples in which the activity is very low or the amount of material available is limited.

Comparison of the rates of repair of O6-alkylguanines in DNA by rat liver and bacterial O6-alkylguanine-DNA alkyltransferase.

The rates of loss of O6-methylguanine and O6-ethylguanine from rat liver DNA were determined over a time period of 15 min to 4 hr after various doses (5 micrograms/kg to 2 mg/kg) of dimethylnitrosamine and diethylnitrosamine which produced total amounts of these adducts in the range of 300 to 16,000 molecules/cell. This amount is considerably less than the content of O6-alkylguanine-DNA alkyltransferase protein (approximately 60,000 molecules/hepatocyte), and during the time period studied, the adducts were found to be lost with pseudo-first order kinetics. The half-life for O6-methylguanine was 47 min. O6-Ethylguanine was removed 3.6 times more slowly with a half-life of 172 min. The ability of partially purified rat liver O6-alkylguanine-DNA alkyltransferase to remove O6-methylguanine and O6-ethylguanine from [3H]alkyl-labeled DNA substrates in vitro was measured, and it was found that O6-methylguanine was removed 3.4 times more rapidly than was O6-ethylguanine. These results are consistent with the hypothesis that most, if not all, of the repair of these adducts which occurs within the first 4 hr after treatment is due to the alkyltransferase protein. Diethylnitrosamine, which is slightly more potent as a carcinogen to rat liver, produced a total amount of O6-ethylguanine of 3.7 mumol/mol guanine/mg compared to O6-methylguanine (28 mumol/mol guanine/mg) given by dimethylnitrosamine. The slower rate of loss of the ethyl adduct is not sufficient to account for this difference, and the results, therefore, support the concept that other DNA adducts (possibly O-alkylpyrimidines) contribute to the initiation of tumors by diethylnitrosamine. Preliminary evidence that the rat liver alkyltransferase can also remove hydroxyethyl groups from DNA at a rate slower than removal of ethyl groups was also obtained. Bacterial O6-alkylguanine-DNA alkyltransferase was shown to remove methyl, ethyl, and hydroxyethyl groups from the O6 position of guanine in DNA using fluorescence detection to quantitate these adducts. The bacterial protein removed methyl groups very rapidly but was much slower than the rat liver protein on the larger adducts. These results suggest that the relative rates of repair of different alkyl groups may be species specific and must be determined experimentally in the cell of interest before conclusions concerning biological effects can be drawn.

Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo[a]pyrenediol epoxide-modified DNA.

DNA replication and transcription are affected adversely by the presence of bulky adducts that are generated by the covalent binding of a variety of metabolically activated environmental pollutants to cellular DNA. When these lesions are not cleared by cellular repair enzymes prior to replication, mutations and ultimately tumor initiation can occur. Transcription and DNA repair appear to be intimately connected, since certain adducts are more efficiently removed from the transcribed strands of active loci than from non-transcribed strands and other quiescent domains in the genome. The mechanism by which RNA polymerases deal with bulky adducts during DNA transcription is therefore of great interest. The availability of site-specifically modified and stereochemically defined oligodeoxyribonucleotides derived from the covalent reaction of 7r, 8t-dihydroxy-9, 10t-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with guanine residues prompted us to study the efficiencies of transcription past these lesions using bacteriophage T7 RNA polymerase. We show here that T7 RNA polymerase can bypass such lesions in a DNA template, providing that a cytosine residue is incorporated opposite anti-BPDE-modified guanine. However, when an incorrect base (most frequently a purine) is inserted opposite the modified site, the RNA polymerase stalls, and the complex dissociates, resulting in a truncated transcript. The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mechanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template.

Intragenomic repair heterogeneity of DNA damage.

The mutagenic and carcinogenic consequences of unrepaired DNA damage depend upon its precise location with respect to the relevant genomic sites. Therefore, it is important to learn the fine structure of DNA damage, in particular, proto-oncogenes, tumor-suppressor genes, and other DNA sequences implicated in tumorigenesis. Both the introduction and the repair of many types of DNA lesions are heterogeneous with respect to chromatin structure and/or gene activity. For example, cyclobutane pyrimidine dimers are removed more efficiently from the transcribed than the nontranscribed strand of the dhfr gene in Chinese hamster ovary cells. In contrast, preferential strand repair of alkali-labile sites is not found at this locus. In mouse 3T3 cells, dimers are more efficiently removed from an expressed proto-oncogene than from a silent one. Persistent damage in nontranscribed domains may account for genomic instability in those regions, particularly during cell proliferation as lesions are encountered by replication forks. The preferential repair of certain lesions in the transcribed strands of active genes results in a bias toward mutagenesis owing to persistent lesions in the nontranscribed strands. Risk assessment in environmental genetic toxicology requires assays that determine effective levels of DNA damage of producing malignancy. The existence of nonrandom repair in the mammalian genome casts doubt on the reliability of overall indicators of carcinogen-DNA binding and lesion repair for such determinations. Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response to particular environmental agents.

Transcription and DNA damage: a link to a kink.

Living organisms are constantly exposed to a variety of naturally occurring and man-made chemical and physical agents that pose threats to health by causing cancer and other illnesses, as well as cell death. One mechanism by which these moieties can exert their toxic effects is by inducing modifications to the genome. Such changes in DNA often result in the formation of nucleotides not normally found in the double helix, bases containing covalent chemical alterations, single- and double-strand breaks, and interstrand and intrastrand cross-links. When these lesions are present during replication, mutations often result in the newly synthesized DNA. Likewise, when such damage occurs in a gene, transcription elongation, and hence expression, can be adversely affected because of pausing or arresting of the RNA polymerase at or near the altered site; this could result in the synthesis of a defective RNA molecule. It has become increasingly clear that transcription and DNA damage are intimately linked, since the removal of certain adducts from the genome is highly dependent on their location. When such lesions are present on the transcribed strand of actively expressed genetic loci, they are better cleared from that strand when compared to the complementary DNA or other quiescent regions. This process is called transcription-coupled DNA repair, and it modulates the mutagenic spectrum of many DNA-damaging agents. Furthermore, based upon evidence from systems in which it is absent, this process has a profound effect on ameliorating the adverse consequences of exposure to many environmentally relevant genotoxins. The precise cellular pathway that mediates the preferential clearance of DNA damage from active genetic loci has not yet been established, but it appears to be effected by a repertoire of proteins that are also involved in other DNA repair pathways and transcription as well as some factors that might be unique to it. Because a cellular process as indispensable as gene expression can be thwarted by the presence of DNA damage, an understanding of the mechanism underlying transcription-coupled DNA repair is relevant to the continued discernment of how environmental genotoxins endanger human health.

Studies of the repair of O6-alkylguanine and O4-alkylthymine in DNA by alkyltransferases from mammalian cells and bacteria.

O6-Methylguanine in DNA is repaired by the action of a protein termed O6-alkylguanine-DNA alkyltransferase (AT) which transfers the methyl group to a cysteine residue in its own sequence. Since the cysteine which is methylated is not regenerated rapidly, if at all, the capacity for repair of O6-methylguanine is limited by the number of molecules of the AT available within the cell. The level and inducibility of the AT differed greatly in different mammalian cell types and species with the highest levels in human tissues and in liver and the lowest levels in brain. Only a small induction occurred in rat liver in response to exposure to alkylating agents. In E. coli such exposure increased the activity more than 100-fold. The At was not specific for methyl groups but also removed ethyl, 2-hydroxyethyl, n-propyl, isopropyl and n-butyl groups from the O6-position in DNA. The protein isolated from E. coli removed methyl groups much more rapidly than the larger alkyl groups but the mammalian AT isolated from rat liver showed much less difference in rate with adducts of different size. Ethyl and n-propyl groups were removed by the rat liver AT only three to four times more slowly than methyl groups. Another important difference between the bacterial and mammalian ATs is that the bacterial protein was also able to remove methyl groups from the O4-position of thymine in methylated DNA or poly(dT) but the AT from rat liver or human fibroblasts did not repair O4-methylthymidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of O6-alkylguanine-DNA-alkyltransferase by metals.

The activity of the DNA-repair protein O6-alkylguanine-DNA-alkyltransferase was found to be strongly inhibited by a number of metal ions. Cd2+ was the most active followed by Cu2+, Hg2+, Zn2+ and Ag2. This inhibition is likely to result from the interaction of the metals with the cysteine-acceptor residue on the protein since the inhibition was reduced by increasing the concentration of dithiothreitol in the assay buffer. These results raise the possibility that exposure to Cd2+ could increase the mutagenicity and carcinogenicity of alkylating agents by retarding the rate of repair of alkylated DNA. However, other metals or metallic compounds which are known to be carcinogenic (such as compounds containing arsenic, lead, nickel or chromium) did not interfere with DNA repair by this protein.

Lack of sequence-specific removal of N-methylpurines from cellular DNA.

The removal of N-methylpurines from the DHFR gene and an unexpressed adjacent locus located downstream occurs at similar rates and to a similar extent in dimethyl sulfate treated Chinese hamster ovary B11 cells. Furthermore, no significant differences in repair rates are observed between the strands of the active gene. These data primarily reflect the removal of the most abundant lesion produced by dimethyl sulfate, 7-methylguanine, and are in contrast to the results obtained for the removal of ultraviolet-induced cyclobutane pyrimidine dimers from the same region of the genome. Pyrimidine dimers are cleared preferentially from the transcribed strand of the DHFR gene and are removed poorly from the non-transcribed complementary strand and unexpressed adjacent regions. The results suggest that DNA lesions such as dimers that block transcription are removed preferentially from active genes, whereas lesions that do not interfere with nucleic acid synthesis (i.e. 7-methylguanine) are removed at similar rates from expressed and silent loci.

Two expressed human genes sustain slightly more DNA damage after alkylating agent treatment than an inactive gene.

Alkylating agent damage was quantified in human T-lymphocytes by calculating gene-specific lesion frequencies and repair rates. At 3 time points after exposure to methyl methanesulfonate (0, 6, and 24 h), T-lymphocyte DNA was extracted, digested with HindIII, and divided into 2 aliquots. Apurinic sites were formed in the DNA fragments of both aliquots by heat-induced liberation of the N-methylpurines. The methoxyamine-treated aliquot provided gene fragments which were refractory to alkaline hydrolysis (full-length fragments), while the fragments in the untreated aliquot were cleaved at apurinic sites by hydroxide. After Southern blotting, lesion frequencies were calculated by comparing the band intensity of the full-length fragment to its unprotected counterpart. The restriction fragments analyzed were from the constitutively active dihydrofolate reductase (dhfr) plus hypoxanthine phosphoribosyltransferase (hprt) genes and from the transcriptionally inactive Duchenne muscular dystrophy gene (dmd). In decreasing order, the fragments containing the most lesions per kb of DNA were: hprt greater than dhfr greater than dmd. T-Lymphocytes from 2 females had 30% more heat-labile N-methylpurines in the active X-linked hprt gene than in the inactive X-linked dmd gene. The lesion frequency found in the male's lone hprt allele was the highest observed. These lesion frequency differences are discussed in terms of chromatin structure. After 6 and 24 h, no significant repair rate differences were observed among the 3 genes.

[Inflammatory pseudotumor of the lung. Diagnostic-therapeutic effectiveness of its radical resection]. / Pseudotumore infiammatorio del polmone. Utilità diagnostico-terapeutica della resezione radicale.

Clinical, therapeutical observations and experience in 3 cases of pulmonary inflammatory pseudotumors (PIP) are presented. A retrospective analysis is made of cases with pulmonary "mass" suspected as malignant tumor, resected in a general surgery department between 1988 and 1995, and finally diagnosed as inflammatory pseudotumor. Three of the 10 cases originally diagnosed as malignant lung tumor were inflammatory pseudotumor (30%). Pulmonary inflammatory pseudotumors, may be a pitfall diagnosing a lung mass and implicate legal problems. Surgical resection leads to the final diagnosis in doubtful cases. A wide resection has a diagnostic aim and may preserve healthy parenchyma. Clinicians, pathologists and surgeons should accurately inform patients with doubtful diagnosis of pulmonary malignancy. Any decision should be kept altogether either choosing the simple observation or the timely surgical diagnostic and therapeutical approach.

[Reconversion after Hartmann's procedure. Our experience]. / Ricanalizzazione dopo Hartmann. Nostra esperienza.

An increasing number of intestinal reconversion after Hartmann have been performed in recent years, especially due to improved surgical techniques and progressively lengthened lifespan. The authors report 33 cases of intestinal recanalization of 100 interventions according to Hartmann from 1984 to 1996 (21 not neoplastic pathologies, 12 neoplasias). The variables considered included: patient age, type of disease requiring intervention according to Hartmann, oncologic characteristic of patients with neoplasia, interval between the two interventions, preoperative examinations performed, morbidity and mortality after reconversion. Furthermore, the fundamental indications for reconversion are described, in particular in patients with neoplasias (CEA, transanal echo, total body Ct, anal manometry). The low frequency of preoperative complications, zero mortality, satisfactory long-term follow-up (only one patient with neoplastic relapse) indicate that colon-rectal reconversion can also be performed in the elderly and patients with neoplasias with favorable prognosis.

Repair of N-methylpurines in specific DNA sequences in Chinese hamster ovary cells: absence of strand specificity in the dihydrofolate reductase gene.

We have developed a quantitative method for examining the removal of N-methylpurines from specific genes to investigate their possible differential repair throughout the genome. Chinese hamster ovary cells were exposed to dimethyl sulfate, and the isolated DNA was treated with an appropriate restriction endonuclease. The DNA was heated to convert remaining N-methylpurines to apurinic sites to render them alkaline-labile. Duplicate samples heated in the presence of methoxyamine to protect the apurinic sites from alkaline hydrolysis provided controls to assess total DNA. After alkaline hydrolysis, agarose gel electrophoresis, Southern transfer, and probing for the fragment of interest, the ratios of band intensities of the test DNA sample to its methoxyamine-treated control counterpart were calculated to yield the percentage of fragments containing no alkaline-labile sites. The frequency of N-methylpurines was measured at different times after dimethyl sulfate treatment to study repair. We found no differences between the rates of repair of N-methylpurines in the active dihydrofolate reductase gene and a nontranscribed region located downstream from it in treated cells. Also, similar rates of repair were observed in the transcribed and nontranscribed strands of the gene, in contrast to previous results for the removal of cyclobutane pyrimidine dimers. Thus, there does not appear to be a coupling of N-methylpurine repair to transcription in Chinese hamster ovary cells. However, the repair in the dihydrofolate reductase domain appears to be somewhat more efficient than that in the genome overall. Our method permits the quantifying at the defined gene level of abasic sites or of any DNA adduct that can be converted to them.

3-Methyladenine and 7-methylguanine exhibit no preferential removal from the transcribed strand of the dihydrofolate reductase gene in Chinese hamster ovary B11 cells.

The removal of cylclobutane pyrimidine dimers from cellular DNA occurs preferentially in actively transcribed genes of cells subjected to ultraviolet radiation. In contrast, reports concerning the transcription-dependent repair of N-methylpurines formed in cellular DNA following exposure to methylating agents are quite conflicting, with some studies suggesting that no biased clearance of these lesions occurs and others indicating that preferential removal of these adducts transpires in active genetic loci. Even in the cases where no preferential clearance was demonstrated, a slight but statistically insignificant biased removal of N-methylpurines from the transcribed strand of active genes was often evident. We proposed that these results might be due to the preferential clearance of only one of the two principal N-methylpurines formed, 3-methyladenine, or to the source of the methylating species to which the cells were exposed. Therefore, we investigated the clearance of 3-methyladenine and 7-methylguanine as individual lesions from the amplified dihydrofolate reductase gene of Chinese hamster ovary cells, and we examined the gene-specific removal of N-methylpurines formed by several different methylating agents as well. We observed no biased clearance of 3-methyladenine toward the transcribed strand of the locus being examined. This result indicates that any minor gene-specific preferential repair that has been observed previously for N-methylpurines in toto--which actually reflects the removal of the predominant methylated purine 7-methylguanine--is not due to biased clearance of the transcription-inhibiting 3-methyladenine lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Repair of O6-propylguanine and O6-butylguanine in DNA by O6-alkylguanine-DNA alkyltransferases from rat liver and E. coli.

DNA substrates containing O6-n-butylguanine, O6-iso-butylguanine, O6-n-propylguanine and O6-iso-propylguanine were prepared by reaction of calf thymus DNA with the appropriate N-alkyl-N-nitrosourea. These substrates were used to test the ability of O6-alkylguanine-DNA alkyltransferases from Escherichia coli and rat liver to remove such alkyl groups from the O6-position of guanine. It was found that all of these adducts were removed by the alkyltransferases, but the branched alkyl chain iso-butyl- and iso-propyl adducts were removed very slowly. Also, when tested with a DNA substrate containing both O6-n-propylguanine and O6-iso-propylguanine, the alkyltransferases removed almost all of the n-propyl-adduct before the iso-propyl-adduct was attacked. Both alkyltransferases showed a decreasing rate of reaction as the size of the alkyl group increased, but there was a significant difference between the rat liver and E. coli alkyltransferase in the relative rates. The rat liver alkyltransferase repaired O6-methylguanine more slowly than the E. coli protein, but was considerably more rapid than the bacterial equivalent when acting on n-propyl- and n-butyl-adducts. The relative rates of repair were methyl much greater than ethyl greater than n-propyl greater than n-butyl greater than iso-propyl, iso-butyl for the E. coli alkyl-transferase and methyl greater than ethyl, n-propyl greater than n-butyl greater than iso-propyl, iso-butyl greater than 2-hydroxyethyl for the rat liver protein. These results indicate that differential rates of repair may contribute to the relative risks of carcinogenesis and mutagenesis by exposure to alkylating agents of different size and that rates of repair may be species specific and must be determined from specific measurements rather than extrapolated from data on other organisms.

Comparison of repair of methylated pyrimidines in poly(dT) by extracts from rat liver and Escherichia coli.

Partially purified preparations of O6-alkylguanine-DNA alkyltransferase from rat liver and E. coli were tested for their ability to repair O4-methylthymine in a methylated poly(dT) X poly(dA) substrate. The bacterial preparation readily carried out this reaction, but no loss of O4-methylthymine was obtained with the rat liver protein. These results indicate a significant difference in specificity between the mammalian and bacterial proteins which could have important consequences for carcinogenesis and mutagenesis by alkylating agents in mammalian cells.

Bacteriophage T7 RNA polymerase transcription elongation is inhibited by site-specific, stereospecific benzo[c]phenanthrene diol epoxide DNA lesions.

Benzo[c]phenanthrene diol epoxide (B[c]PhDE), the ultimate carcinogenic metabolite of the environmental pollutant benzo[c]phenanthrene, reacts with DNA primarily at the exocyclic amino groups of purines, forming B[c]PhDE-DNA adducts that differ in their stereochemical configurations and their effect on biological processes such as transcription. To determine the effect of these stereoisomers on RNA synthesis, in vitro T7 RNA polymerase transcription assays were performed using DNA templates modified on the transcribed strand by either a site-specific (+)-trans- or (-)-trans-anti-B[c]PhDE-N(6)-dA lesion located within the sequence 5'-CTCTCACTTCC-3'. The results show that both (-)-trans-anti-B[c]PhDE-N(6)-dA and (+)-trans-anti-B[c]PhDE-N(6)-dA block RNA synthesis. Furthermore, both B[c]PhDE-dA stereoisomeric adducts lead to lower levels of initiation of transcription relative to that observed using an unmodified DNA template. In contrast to these results, placement of the adduct on the nontranscribed strand within the template does not impede transcription elongation. In addition to the assessment of the effect of the lesions on transcription elongation, the resulting transcripts were characterized in terms of their base composition. A high level of base misincorporation is detected at the 3'-ends of truncated transcripts, with guanosine being most frequently incorporated opposite the modified nucleotide rather than the expected uridine. This result supports the notion that translocation past a modified base in a DNA template relies in part on correct base incorporation, and suggests that stalling of RNA polymerases at damaged sites in DNA may well be dependent on both the presence of the lesion and the base which is incorporated opposite the modified nucleotide.

Site-specific benzo[a]pyrene diol epoxide-DNA adducts inhibit transcription elongation by bacteriophage T7 RNA polymerase.

Benzo[a]pyrene, an extremely potent procarcinogen and mutagen, is metabolized to a variety of products, including the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene. This product of biotransformation reacts with DNA, forming a series of adducts principally at the N2 position of guanine that differ in their stereochemistry and exhibit unique biological properties. In order to gain a better understanding of the effects on RNA synthesis of these adducts, we used purified bacteriophage T7 RNA polymerase to transcribe a series of templates containing one of four stereoisomerically pure BPDE-guanine lesions--(+)-trans-,(-)-trans-,(+)-cis-anti-N2-BPDE-guanine--or no damaged bases. To construct suitable double-stranded oligodeoxynucleotides for these studies, we annealed an 11-mer containing a site-specific stereoisomerically pure N2-BPDE-guanine adduct, a 37-mer, and a 10-mer to a complementary 58-base sequence of single-stranded DNA. The oligomers were ligated, purified, and reannealed. The resulting DNA template contained the promoter for T7 RNA polymerase and a BPDE adduct at position +16 following the transcription initiation site. The results of the transcription assays clearly demonstrate that each of the adducts inhibits elongation by T7 RNA polymerase, but they do so to significantly different extents, depending on the stereochemical characteristics of the BPDE-modified guanine. The order of inhibition is (+)-trans > (-)-trans > (+)-cis > (-)-cis, when the amount of full-length transcript for each is compared to that obtained for an unmodified template. Furthermore, premature termination of RNA synthesis occurs at or near the site of the BPDE lesion as evidenced by the formation of discrete, truncated transcripts. These results might be related to the fact that the pyrenyl moiety of the trans-BPDE adducts is situated in the minor groove of double-stranded DNA, but is quasi-intercalated into the double helix in the case of the cis stereoisomers.(ABSTRACT TRUNCATED AT 250 WORDS)