PARP-2 sustains erythropoiesis in mice by limiting replicative stress in erythroid progenitors.

Erythropoiesis is a tightly regulated process in which multipotential hematopoietic stem cells produce mature red blood cells. Here we show that deletion of poly(ADP-ribose) polymerase-2 (PARP-2) in mice leads to chronic anemia at steady state, despite increased erythropoietin plasma levels, a phenomenon not observed in mice lacking PARP-1. Loss of PARP-2 causes shortened lifespan of erythrocytes and impaired differentiation of erythroid progenitors. In erythroblasts, PARP-2 deficiency triggers replicative stress, as indicated by the presence of micronuclei, the accumulation of γ-H2AX (phospho-histone H2AX) in S-phase cells and constitutive CHK1 and replication protein A phosphorylation. Transcriptome analyses revealed the activation of the p53-dependent DNA-damage response pathways in PARP-2-deficient cells, culminating in the upregulation of cell-cycle and cell death regulators, concomitant with G2/M arrest and apoptosis. Strikingly, while loss of the proapoptotic p53 target gene Puma restored hematocrit levels in the PARP-2-deficient mice, loss of the cell-cycle regulator and CDK inhibitor p21 leads to perinatal death by exacerbating impaired fetal liver erythropoiesis in PARP-2-deficient embryos. Although the anemia displayed by PARP-2-deficient mice is compatible with life, mice die rapidly when exposed to stress-induced enhanced hemolysis. Our results pinpoint an essential role for PARP-2 in erythropoiesis by limiting replicative stress that becomes essential in the absence of p21 and in the context of enhanced hemolysis, highlighting the potential effect that might arise from the design and use of PARP inhibitors that specifically inactivate PARP proteins.

Loss of poly(ADP-ribose) polymerase-2 leads to rapid development of spontaneous T-cell lymphomas in p53-deficient mice.

Poly(ADP-ribose) polymerase-2 (Parp-2) belongs to a family of enzymes that catalyse poly(ADP-ribosyl)ation of proteins. Parp-2 deficiency in mice (Parp-2(-/-)) results in reduced thymic cellularity associated with increased apoptosis in thymocytes, defining Parp-2 as an important mediator of T-cell survival during thymopoiesis. To determine whether there is a link between Parp-2 and the p53 DNA-damage-dependent apoptotic response, we have generated Parp-2/p53-double-null mutant mice. We found that p53(-/-) backgrounds completely restored the survival and development of Parp-2(-/-) thymocytes. However, Parp-2-deficient thymocytes accumulated high levels of DNA double-strand breaks (DSB), independently of the p53 status, in line with a function of Parp-2 as a caretaker promoting genomic stability during thymocytes development. Although Parp-2(-/-) mice do not have spontaneous tumours, Parp-2 deficiency accelerated spontaneous tumour development in p53-null mice, mainly T-cell lymphomas. These data suggest a synergistic interaction between Parp-2 and p53 in tumour suppression through the role of Parp-2 in DNA-damage response and genome integrity surveillance, and point to the potential importance of examining human tumours for the status of both genes.

SHV-1 beta-lactamase is mainly a chromosomally encoded species-specific enzyme in Klebsiella pneumoniae.

The nature of the SHV-1 beta-lactamase gene was analyzed in 97 epidemiologically unrelated Klebsiella pneumoniae strains isolated from clinical samples. beta-Lactamase bands that focused at a pI of 7.6 (SHV-1-type) in 74 strains, at a pI of 7.1 (LEN-1-type) in 13 strains, and at a pI of 5.4 (TEM-1-type) in 10 strains were detected by analytical isoelectric focusing (IEF). Among the 74 SHV-1-producing strains, 40 had, in addition to the pI 7.6 band, an additional band on IEF: 20 had a band with a pI of 7.1 and 20 had a band with a pI of 5.4. Most of the 74 SHV-1-producing strains (76.7%) carried plasmids. Transfer of beta-lactam resistance by conjugation was possible in only 9.3% of the strains tested. SHV-1 gene-specific PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the chromosomal DNA was positive for 93 of the 97 strains and negative for only 4 of the 10 samples with K. pneumoniae TEM-1 producers. In an attempt to approximate the location of the SHV gene locus by endonuclease restriction analysis, RFLP analysis with Southern blotting of chromosomal DNA with a labeled SHV-1 fragment as a probe was used to study the 97 strains. A trial with EcoRI showed at least one positive hybridization band for 96 strains; two bands were detected for 8 strains. The hybridization was negative for only one TEM-1 beta-lactamase-producing strain. DNA sequence analysis showed no differences in promoter regions or extra stop-triplet sequences; only point mutations determined different allelic variants. The novel SHV-type variants are designated SHV-32 and SHV-33. As a result of the RFLP and sequencing analyses, it can be postulated that the loci for SHV-1 and LEN-1 genes are arranged in tandem. Our results strongly support the hypothesis that the ancestor of the SHV-1 beta-lactamase originated from the K. pneumoniae chromosome.

Metabolism of (R)- and (S)-3-(phenylamino)propane-1,2-diol in C57BL/6- and A/J-strain mice. Identification of new metabolites with potential toxicological significance to the toxic oil syndrome.

The Toxic Oil Syndrome was a massive food-borne intoxication that occurred in Spain in 1981. Epidemiological studies point to 3-(phenylamino)propane-1,2-diol (PAP) derivatives as the putative toxic agents. We report further identification of metabolites cleared in urine of A/J and C57BL/6 mice in which (R)- and (S)-3-(phenylamino)propane-1,2-diol were administered intraperitoneally. This investigation is an extension of previous studies carried out with the racemic compound [Ladona, M. G., Bujons, J., Messeguer, A., Ampurdanés, C., Morató, A., and Corbella, J. (1999) Chem. Res. Toxicol. 12, 1127-1137]. Both PAP enantiomers were extensively metabolized, and several metabolites were eliminated in urine. The HPLC profiles of the urine samples of both mouse strains treated with each enantiomer were qualitatively similar, but differences were found in a relatively higher proportion of several detected metabolites in mice treated with (R)-PAP compared with those treated with (S)-PAP. The main urine metabolite continues to be 2-hydroxy-3-(phenylamino)propanoic acid (1), which confirms our previous results obtained with rac-PAP. In addition to the detection of other metabolites already reported in our previous paper, interesting evidence is provided on the presence of 4-aminophenol and paracetamol conjugates in the urine samples from both mouse strains. The detection of these metabolites suggests the in vivo formation of quinoneimine PAP derivatives. Indeed, some quinoneimine species (11 and 12), as well as other PAP metabolites (13) that bear modifications in the alkyl chain, have been tentatively identified in mouse urine. These metabolic findings might imply a potential toxicological significance for the Toxic Oil Syndrome.

Pharmacogenetic profile of xenobiotic enzyme metabolism in survivors of the Spanish toxic oil syndrome.

In 1981, the Spanish toxic oil syndrome (TOS) affected more than 20,000 people, and over 300 deaths were registered. Assessment of genetic polymorphisms on xenobiotic metabolism would indicate the potential metabolic capacity of the victims at the time of the disaster. Thus, impaired metabolic pathways may have contributed to the clearance of the toxicant(s) leading to a low detoxification or accumulation of toxic metabolites contributing to the disease. We conducted a matched case-control study using 72 cases (54 females, 18 males) registered in the Official Census of Affected Patients maintained by the Spanish government. Controls were nonaffected siblings (n =72) living in the same household in 1981 and nonaffected nonrelatives (n = 70) living in the neighborhood at that time, with no ties to TOS. Genotype analyses were performed to assess the metabolic capacity of phase I [cytochrome P450 1A1 (CYP1A1), CYP2D6] and phase II [arylamine N-acetyltransferase-2 (NAT2), GSTM1 (glutathione S-transferase M1) and GSTT1] enzyme polymorphisms. The degree of association of the five metabolic pathways was estimated by calculating their odds ratios (ORs) using conditional logistic regression analysis. In the final model, cases compared with siblings (72 pairs) showed no differences either in CYP2D6 or CYP1A1 polymorphisms, or in conjugation enzyme polymorphisms, whereas cases compared with the unrelated controls (70 pairs) showed an increase in NAT2 defective alleles [OR = 6.96, 95% confidence interval (CI), 1.46-33.20] adjusted by age and sex. Glutathione transferase genetic polymorphisms (GSTM1, GSTT1) showed no association with cases compared with their siblings or unrelated controls. These findings suggest a possible role of impaired acetylation mediating susceptibility in TOS.

Metabolism of R,S enantiomers of 3-phenylamino-1,2-propanediol, a compound associated with the toxic oil syndrome, in C57BL/6- and A/J-strain mice.

PAP, a very polar substance, is highly metabolized in mice and excreted principally in urine in the form of the 2-hydroxy-3-phenylaminopropanoic acid of each enantiomer. Thus, the major route of PAP elimination in these strains is alkyl chain oxidation. In particular, S-PAP is eliminated principally in the form of that metabolite, whereas R-PAP enantiomer showed further oxidized species at the aromatic ring and alkyl chain, yielding potential decarboxylated compounds and iminoquinones. All these metabolites may have toxicologic implications. On the other hand, OOPAP intestinal hydrolysis in favour of one PAP enantiomer might be expected since lipases show chiral hydrolysis (unpublished data, manuscript in preparation). In this respect, enantiomeric distribution and metabolic differences should be taken into account in the toxicokinetics of these compounds and their potential association with Toxic Oil Syndrome symptoms.

Biotransformation and clearance of 3-(phenylamino)propane-1,2-diol, a compound present in samples related to toxic oil syndrome, in C57BL/6 and A/J mice.

In May 1981, a massive food-borne intoxication occurred in Spain. The so-called toxic oil syndrome (TOS) was associated with the consumption of aniline-denatured and refined rapeseed oil that was illegally sold as edible olive oil. Fatty acid anilides and fatty acid derivatives of 3-(phenylamino)propane-1,2-diol were detected in oils and implicated as potential toxic agents and markers of toxic oil batches. Epidemiological evidence points to 3-(phenylamino)propane-1,2-diol derivatives as the putative toxic agents, which were generated during the refining process at the ITH refinery. Here we present the biotransformation and clearance of 3-(phenylamino)propane-1,2-diol (PAP) administered intraperitoneally to A/J and C57BL/6 mice that have been proposed as a murine model for the immunological features of TOS. Mice eliminated 6 microCi of [U-(14)C]PAP during a 24 h period, mostly in urine. Animals exhibited urine elimination rates of 70 and 36% in A/J and C57BL/6 strains, respectively. A/J mice exhibited no increase in the elimination rate when induced with beta-naphthoflavone, whereas C57BL/6 did increase the rate of elimination to 57%. Feces contributed to a lesser extent to the elimination rate (0.6 and 3.3% in A/J and C57BL/6 mice, respectively). Radioactivity remaining in organ tissues was lower than 1% (liver, lung, kidney, spleen, heart, and muscle). Metabolic species in urine were identified by HPLC coupled to UV and radioisotope detectors and further GC/MS analyses. 2-Hydroxy-3-(phenylamino)propanoic acid metabolite was the major chemical species excreted in urine in both strains, in both control and induced animal groups. This compound was the main urinary metabolite of PAP, and unmetabolized PAP excreted in urine constituted less than 1% of the total administered dose. Two additional highly polar metabolites also detected in urine were identified as 3-[(4'-hydroxyphenyl)amino]propane-1,2-diol and 2-hydroxy-3-[(4'-hydroxyphenyl)amino]propanoic acid. These findings are the first reported on PAP metabolism and clearance in mice strains and suggest that PAP can be extensively metabolized in vivo and potential reactive species can be generated.