A signature of renal stress resistance induced by short-term dietary restriction, fasting, and protein restriction.

During kidney transplantation, ischemia-reperfusion injury (IRI) induces oxidative stress. Short-term preoperative 30% dietary restriction (DR) and 3-day fasting protect against renal IRI. We investigated the contribution of macronutrients to this protection on both phenotypical and transcriptional levels. Male C57BL/6 mice were fed control food ad libitum, underwent two weeks of 30%DR, 3-day fasting, or received a protein-, carbohydrate- or fat-free diet for various periods of time. After completion of each diet, renal gene expression was investigated using microarrays. After induction of renal IRI by clamping the renal pedicles, animals were monitored seven days postoperatively for signs of IRI. In addition to 3-day fasting and two weeks 30%DR, three days of a protein-free diet protected against renal IRI as well, whereas the other diets did not. Gene expression patterns significantly overlapped between all diets except the fat-free diet. Detailed meta-analysis showed involvement of nuclear receptor signaling via transcription factors, including FOXO3, HNF4A and HMGA1. In conclusion, three days of a protein-free diet is sufficient to induce protection against renal IRI similar to 3-day fasting and two weeks of 30%DR. The elucidated network of common protective pathways and transcription factors further improves our mechanistic insight into the increased stress resistance induced by short-term DR.

Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Genotoxic exposure: novel cause of selection for a functional ΔN-p53 isoform.

The p53 gene is frequently mutated in cancers and it is vital for cell cycle control, homeostasis and carcinogenesis. We describe a novel p53 mutational spectrum, different to those generally observed in human and murine tumors. Our study shows a high prevalence of nonsense mutations in the p53 N terminus of 2-acetylaminofluorene (2-AAF)-induced urinary bladder tumors. These nonsense mutations forced downstream translation initiation at codon 41 of Trp53, resulting in the aberrant expression of the p53 isoform ΔN-p53 (or p44). We propose a novel mechanism for the origination and the selection for this isoform. We show that chemical exposure can act as a novel cause of selection for this truncated protein. In addition, our data suggest that the occurrence of ΔN-p53 accounts, at least in mice, for a cancer phenotype. We also show that gene expression profiles of embryonic stem (ES) cells carrying the ΔN-p53 isoform in a p53-null background are divergent from p53 knockout ES cells, and therefore postulate that ΔN-p53 itself has functional transcriptional properties.

Iodine deficiency activates antioxidant genes and causes DNA damage in the thyroid gland of rats and mice.

Because thyroid nodules are frequent in areas with iodine deficiency the aim of this study was to characterise molecular events during iodine deficiency that could explain mutagenesis and nodule formation. We therefore studied gene expression of catalytic enzymes prominent for H(2)O(2) detoxification and antioxidative defence, quantified DNA oxidation and damage as well as spontaneous mutation rates (SMR) in mice and rats fed an iodine controlled diet. Antioxidative enzymes such as superoxide dismutase 3, glutathione peroxidase 4 and the peroxiredoxins 3 and 5 showed increased mRNA expression, which indicates increased radical burden that could be the cause of additional oxidized base adducts found in thyroidal genomic DNA in our experiments of iodine deficiency. Furthermore, the uracil content of thyroid DNA was significantly higher in the iodine-deficient compared to the control group. While SMR is very high in the normal thyroid gland it is not changed in experimental iodine deficiency. Our data suggest that iodine restriction causes oxidative stress and DNA modifications. A higher uracil content of the thyroid DNA could be a precondition for C-->T transitions often detected as somatic mutations in nodular thyroid tissue. However, the absence of increased SMR would argue for more efficient DNA repair in response to iodine restriction.

Deoxyribonucleic acid damage and spontaneous mutagenesis in the thyroid gland of rats and mice.

Thyroid tumors are a frequent finding not only in iodine-deficient regions. They are predominantly characterized by somatic genetic changes (e.g. point mutations or rearrangements). Because slow thyroid proliferation is a apparent contradiction to a high frequency of tumor initiation, we characterized mutational events in thyroid. First we studied the frequency of certain base exchanges in somatic TSH receptor (TSHR) mutations and determined the spontaneous mutation rate in thyroid and liver. Then we applied different protocols of the comet assay to quantify genomic DNA damage and conducted immunohistochemistry for 8-oxoguanine as a molecular marker for oxidative stress. Among 184 somatic mutations of the human TSHR found in thyroid tumors, C-->T transitions had a unexpectedly high frequency (>32%). The mutation rate in thyroid is 8-10 times higher than in other organs. The comet assay detected increased levels of oxidized pyrimidine (2- to 3-fold) and purine (2- to 4-fold) in thyroid, compared with liver and lung, and a 1.6-fold increase of oxidized purine, compared with spleen. Immunohistochemistry revealed high levels of 8-oxoguanine in thyroid epithelial cells. We have shown a strikingly high mutation rate in the thyroid. Furthermore, results of the comet assay as well as immunohistochemistry suggest that oxidative DNA modifications are a likely cause of the higher mutation rate. It is possible that free radicals resulting from reactive oxygen species in the thyroid generate mutations more frequently. This is also supported by the spectrum of somatic mutations in the TSHR because more frequent base changes could stem from oxidized base adducts that we detected in the comet assay and with immunohistochemistry.

The relationship between benzo[a]pyrene-induced mutagenesis and carcinogenesis in repair-deficient Cockayne syndrome group B mice.

Cockayne syndrome (CS) patients are deficient in the transcription coupled repair (TCR) subpathway of nucleotide excision repair (NER) but in contrast to xeroderma pigmentosum patients, who have a defect in the global genome repair subpathway of NER, CS patients do not have an elevated cancer incidence. To determine to what extent a TCR deficiency affects carcinogen-induced mutagenesis and carcinogenesis, CS group B correcting gene (CSB)-deficient mice were treated with the genotoxic carcinogen benzo(a)pyrene (B[a]P) at an oral dose of 13 mg/kg body weight, three times a week. At different time points, mutant frequencies at the inactive lacZ gene (in spleen, liver, and lung) as well as at the active hypoxanthine phosphoribosyltransferase (Hprt) gene (in spleen) were determined to compare mutagenesis at inactive versus active genes. B[a]P treatment gave rise to increased mutant frequencies at lacZ in all of the organs tested without a significant difference between CSB-/- and wild-type mice, whereas B[a]P-induced Hprt mutant frequencies in splenic T-lymphocytes were significantly more enhanced in CSB-/- mice than in control mice. The sequence data obtained from Hprt mutants indicate that B[a]P adducts at guanine residues were preferentially removed from the transcribed strand of the Hprt gene in control mice but not in CSB-/- mice. On oral treatment with B[a]P, the tumor incidence increased in both wild-type and CSB-deficient animals. However, no differences in tumor rate were observed between TCR-deficient CSB-/- mice and wild-type mice, which is in line with the normal cancer susceptibility of CS patients. The mutagenic response at lacZ, in contrast to Hprt, correlated well with the cancer incidence in CSB-/- mice after B[a]P treatment, which suggests that mutations in the bulk of the DNA (inactive genes) are a better predictive marker for carcinogen-induced tumorigenesis than mutations in genes that are actively transcribed. Thus, the global genome repair pathway of NER appears to play an important role in the prevention of cancer.

Effect of heterozygous loss of p53 on benzo[a]pyrene-induced mutations and tumors in DNA repair-deficient XPA mice.

XPA-deficient mice have a complete deficiency in nucleotide excision repair, and as such they display a cancer predisposition after exposure to several carcinogens. Besides being sensitive to genotoxic agents applied to the skin, they are also susceptible to human carcinogens given orally, like benzo[a]pyrene (B[a]P). To study the role of the tumor suppressor gene p53 in DNA repair, gene mutation, and tumor induction, we crossed XPA-deficient mice with p53 knockout mice and lacZ (pUR288) gene marker mice. When treated orally (by gavage) with B[a]P, the XPA(-/-)/p53(+/-) double transgenic mice developed tumors much earlier and with higher frequency compared to their single transgenic counterparts. The major tumor type found in all genotypes was generalized lymphoma mainly residing in the spleen; several sarcomas were observed in p53(+/-) and XPA(-/-)/p53(+/-) mice. Next, we determined lacZ mutation frequencies in several (non)target tissues. It appeared that in the spleen (the major tumor target tissue) of XPA(-/-) and XPA(-/-)/p53(+/-) mice the lacZ mutation frequency was significantly elevated (80-100 x 10(-5)), and was two times higher as found in spleens of B[a]P-treated WT and p53(+/-) mice (P = 0.003). In nontumor target tissues like liver and lung, we found a moderate increase in the lacZ gene mutation frequency (30-40 x 10(-5)), which was independent of the genotype. The results obtained with the DNA-repair deficient XPA mice indicate that a significantly increased lacZ mutation frequency in a particular organ/tissue is an early marker for tumor development at later stages at the same site. However, the synergistic effect of a XPA(-/-)- and a p53(+/-)-deficiency in tumor development is not reflected by an absolute increase in the lacZ mutation frequency in the major tumor target tissue of XPA(-/-)/p53(+/-) or p53(+/-) mice compared to that of XPA(-/-) and WT mice, respectively.

Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.

Elevated frequencies of benzo(a)pyrene-induced Hprt mutations in internal tissue of XPA-deficient mice.

Xeroderma pigmentosum (XP) patients are hypersensitive to sunlight and have a high predisposition to developing cancer. At the cellular level, XP patients are defective in nucleotide excision repair (NER). Recently, mice have been generated via gene targeting that are deficient in the expression of the XPA gene [A. de Vries et al., Nature (Lond.), 377: 169-173, 1995]. We have assessed the consequences of defective NER for mutagenesis in normal and XPA mice exposed to benzo(a)pyrene and 2-acetylaminofluorene. To study mutagenesis, mature T lymphocytes were isolated from the spleen and stimulated to proliferate in vitro to select for mutants at the endogenous Hprt locus. Background mutant frequencies in normal and XPA mice were very similar and not influenced by age. Single doses of benzo(a)pyrene administered i.p. resulted in a dose-dependent increase of the Hprt mutant frequency in normal mice. In addition, after chronic exposure to benzo(a)pyrene, Hprt mutants were readily detectable in XPA mice at an early onset of treatment but only at a later stage in normal mice. In contrast, chronic treatment of either normal or XPA mice with 2-acetylaminofluorene did not increase Hprt mutant frequency above the background frequency. This absence of significant induction of Hprt mutants can be entirely attributed to the low frequency of 2-acetylaminofluorene-induced DNA adducts in lymphoid tissue. These results provide the first direct evidence in mammals that deficient NER leads to enhanced mutagenesis in endogenous genes in internal tissue after exposure to relevant environmental mutagens, such as benzo(a)pyrene.

Spontaneous liver tumors and benzo[a]pyrene-induced lymphomas in XPA-deficient mice.

Defects in the xeroderma pigmentosum complementation group A-correcting (XPA) gene, which encodes a component of the nucleotide excision repair (NER) pathway, are associated with the cancer-prone human disease xeroderma pigmentosum. We previously generated mice lacking the XPA gene, which develop normally but are highly sensitive to ultraviolet-B and 7,12-dimethylbenz[a] anthracene-induced skin tumors. Here we report that XPA-deficient mice spontaneously developed hepatocellular adenomas at a low frequency as they aged. Furthermore, oral treatment of XPA-deficient mice with the carcinogen benzo[a]pyrene (B[a]P) resulted in the induction of mainly lymphomas. These tumors appeared earlier and with a higher incidence than in B[a]P-treated wild-type and heterozygous mice. Our results show for the first time that XPA-deficient mice also displayed an increased sensitivity to developing tumors other than tumors of the skin.

Use of E mu-PIM-1 transgenic mice short-term in vivo carcinogenicity testing: lymphoma induction by benzo[a]pyrene, but not by TPA.

E mu-pim-1 transgenic mice are predisposed to develop lymphomas. Due to their low spontaneous tumour incidence and their increased sensitivity towards the lymphomagen ethylnitrosourea these mice may present an interesting model for short-term carcinogenicity testing. Here, we report on the further exploration of this transgenic mouse model with two additional carcinogens known to have, among others, the lymphohaematopoietic system as target, i.e. benzo[a]pyrene (B[a]P) and 12-O-tetradecanoylphorbol-13-acetate (TPA). B[a]P, given three times a week (by gavage) for 13 weeks at 4.3, 13 or 39 mg/kg body weight, resulted in a dose-related increase in lymphomas up to a 90% incidence in E(mu)-pim-1 mice during the observation period of 40 weeks. B[a]P also induced tumours of the forestomach within this observation period, though at a lower incidence and apparently equally effective in wildtype and transgenic mice. TPA, on the other hand, was unable to induce lymphomas (or tumours in any other organ) in either transgenic or wildtype animals within the observation period of 44 weeks, when applied dermally at the maximum tolerated dose of 3 microg/mouse, twice a week for 35 weeks. Molecular analysis showed that B[a]P-induced lymphomas in transgenic mice were of T-cell origin, 80% of which had elevated levels of c-myc expression. None of the lymphomas had increased N-myc expression and mutation analysis of the ras-gene family revealed a K-ras mutation in only one out of eight tumours investigated. Also, none of the lymphomas showed aberrant expression of p53 as determined by immunohistochemistry. It is concluded that the E mu-pim-1 mouse model will not be very suitable for short-term carcinogenicity testing in general: only genotoxic chemicals that have the lymphohaematopoietic system as target for carcinogenesis in wild-type mice, appear to be efficiently identified.

Vascular malformations: a review of 10 years' management in a university hospital.

In order to gain insight into the management of patients with vascular malformations (VM) in the University Hospital Nijmegen in the past 10 years, 151 cases managed by different specialists were reviewed. To avoid the usual confusion in terminology, all recorded diagnoses were reclassified according to the biological classification of Mulliken. The sex distribution was equal; 79% of the malformations were diagnosed at birth or in the 1st year of life. The median time between presentation and consultation was 3 years. Sixty-two lymphatic, 26 venous, 24 capillary, 1 arterial, and 38 combined malformations (8 arteriovenous, 30 others) were found. The head and neck region was most frequently involved, followed by the lower and upper limbs and trunk. The pediatric surgeon was the most frequently consulted specialist. Confusing, mutually incompatible terminology and a wide variety of different diagnostic techniques and treatments had been used by the different specialists. To improve the management of patients with vascular malformations, the use of a uniform classification, an increase in basic investigations, and the development and evaluation of protocols for diagnosis and treatment by multidisciplinary teams are necessary.

Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.

Xeroderma pigmentosum patients with a defect in the nucleotide-excision repair gene XPA are characterized by, for example, a > 1,000-fold higher risk of developing sunlight-induced skin cancer. Nucleotide-excision repair (NER) is involved in the removal of a wide spectrum of DNA lesions. The XPA protein functions in a pre-incision step, the recognition of DNA damage. To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells. The XPA-/-mice appear normal, at least until the age of 13 months. XPA-/-mice are highly susceptible to ultraviolet (UV)-B-induced skin and eye tumours and to 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumours. We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum.

Results of splenectomy performed on a group of 91 children.

In the period 1971-1990 91 children underwent splenectomies in the University Hospital of Nijmegen. The most important indications are hereditary spherocytosis, Hodgkin's disease and very severe immune thrombocytopenic purpura (ITP). Splenectomy after a traumatic rupture of the spleen has become less frequent: from 20% in 1971-1980 to 4% in 1981-1990. Short-term complications included thrombocytosis (84%), fever without an obvious cause (46%), which is quite regularly seen in patients suffering from Hodgkin's disease (48%), and infections of the respiratory tract in 10% of the patients. The platelet count shows a steady increase in the first nine post-operative days. No thromboembolic complications were seen. Based upon the literature there seems to be no reason at this moment for anti-platelet aggregation therapy when platelet counts are below 1000 x 10(9)/l. More information about long-term complications was obtained through a questionnaire completed by general practitioners. The morbidity through overwhelming post splenectomy infection (OPSI) is 3.8% (3/79), the mortality of OPSI is 2.5% (2/79). Underlying diseases, especially those which involve the immunological system as auto immune haemolytic anemia (AIHA), seem to play an important role in the possible development of OPSI (morbidity 2/11, 18%).

Neurological complications in children with protein C deficiency.

Four children with a cerebrovascular occlusive accident and protein C deficiency are described. Two patients presented with an acute hemiplegia, the others suffered from a transient ischemic attack and a progressive hydrocephalus as the result of sinus thrombosis. In all cases protein C deficiency, Type 1, was diagnosed. Other causes of cerebrovascular disease were excluded. Although venous thrombosis has been extensively reported in protein C deficiency, these cases indicate that protein C deficiency is also related to arterial thrombosis. In evaluating children with cerebrovascular accidents, protein C deficiency should also be considered.

Cloning and characterization of the mouse XPAC gene.

Xeroderma Pigmentosum is a human disease, which is, among others, characterized by a high incidence of (sunlight induced) skin cancer, due to a defect in nucleotide excision repair (NER). The human DNA repair gene XPAC corrects this defect in cells isolated from Xeroderma Pigmentosum complementation group A (XP-A) patients. To enable the development of a transgenic mouse model for XP-A by gene targeting in embryonic stem cells, we cloned and characterized the mouse homologue of the XPAC gene. The mouse XPAC gene was found to consist of 6 exons, spanning approximately 21 kb. The nucleotide sequence of the exons is identical to that of the also cloned the mouse XPAC cDNA. Furthermore, the deduced amino acid sequence of the XPAC protein is the same as the one published previously by Tanaka et al. From CAT assay analysis, the promoter of the XPAC gene appeared to be located within 313 bp upstream of the assumed transcriptional start site. Like the promoters of other eukaryotic DNA repair genes (i.e. ERCC-1 and XPBC/ERCC-3), the mouse XPAC promoter region lacks classical promoter elements like TATA-, GC- and CAAT boxes. However, it contains an unique polypyrimidine-rich box, which is so far only found in genes encoding DNA repair enzymes. The function of this box in the regulation of transcription is still unclear.

Vitamin K status in cystic fibrosis.

Appearance of PIVKA-II (protein induced by vitamin K absence-II) in serum is a biochemical sign of insufficient vitamin K-dependent carboxylation of prothrombin. Plasma concentrations of PIVKA-II and vitamin K1 were determined in 24 children with cystic fibrosis. Eight were supplemented with vitamin K1. The purpose of the study was to determine the occurrence of vitamin K deficiency in cystic fibrosis and to evaluate the effect of vitamin K supplementation. PIVKA-II was detectable in only one unsupplemented child. In this patient, the concentration of vitamin K1 was below the limit of detection of 60 ng/l. Vitamin K1 levels in the other unsupplemented children were normal (mean 476 ng/l = 1 mmol/l). The supplemented patients showed extremely high levels of vitamin K1 (mean 22445 ng/l = 50 nmol/l). In conclusion, vitamin K deficiency occurs infrequently in cystic fibrosis. Checking the coagulation system is advised, but routine vitamin K supplementation is not recommended. If additional vitamin K is needed, the starting dose should not exceed 1 mg daily.

Fetal periventricular hemorrhage in von Willebrand's disease: short review and first case presentation.

We present the first case of fetal periventricular hemorrhage due to type IIa Von Willebrand's disease. Discussed are the causal relationships between fetal hemostatic disorders and periventricular hemorrhage, the risks of labor and delivery, and the management with respect to antenatal diagnosis, fetal therapy, and delivery.