32P-Postlabelling analysis of the DNA adducts formed by aristolochic acid I and II

We report the quantitation of DNA adducts in target and non-targetorgans of male Wistar rats treated orally with five daily doses(10 mg/kg body wt) aristolochic acid I (AAI) or aristolochicacid II (AAII), the major components of the herbal drug aristolochicacid, a forestomach carcinogen In the rat. DNA adducts weredetected and analysed using the nuclease P1-enhanced variationof the Randerath 32 postlabeiling assay. The highest level ofDNA adducts formed was by AAI inthe target organ, forestomach(330 ± 30 adducts/10⁸ nucleotides), but high levels werealso observed in a non-target tissue, the glandular stomach(180 ± 15). Lower amounts of adducts were detected inliver, kidney and urinary bladder epltheliuin. With AAII thebinding Levels were generally lower than the AAII, the highestLevel of adducts being detected in kidney (80 ± 20 adducts/10⁸nucleotides) and lower levels in liver, stomach and urinarybladder epithelia. Adduct patterns similar to those in vivowere observed in two new in vitro assays. Rat faecal bacteriawere shown to be able to activate AM and AAII to reactive species,which were trapped with exogenous calf thymus DNA and analysedby postlabelling. llncuhatlon of AM and AAII in explanted ratstomach held in short-term organ culture resulted In DNA adductformation in the epithelia of both forestomach and glandularstomach. To assign the recently characterized in vitro nucleosideadducts of AII to the bisphosphate derivatives, a new ion-pairH]PLC procedure on a reversed-phase column was developed. Bymonitoring Cerenkov radiation on-line, a good separation ofAII adducts was observed, demonstrating that adducts formedin vivo were chromatographically indistinguishable with thoseformed in vitro, and previously characterized as an aristolactammoiety bound covalently to the exocydlic amino groups of deoxyadenosineand deoxyguanosine.     

Thrombotic thrombocytopenic purpura in early pregnancy with maternal and fetal survival

Summary Thrombotic thrombocytopenic purpura (TTP) is a hematologic disorder which is clinically characterized by thrombocytopenia, microangiopathic hemolytic anemia, fever, neurologic symptoms, and cardiac and renal involvement. The pathogenic mechanisms of this disease are poorly understood. It is well known that TTP is associated with pregnancy and that prognosis for the mother and child is poor. We present the first case of a severe TTP diagnosed in the first trimester of pregnancy (13th week of gestation) with maternal survival and birth of a healthy child which required continuous and intensive treatment with plasmatherapy until delivery. During a period of 24 weeks several attempts to discontinue plasma therapy failed because of continuous active disease, and it became evident that plasma infusions were not as effective as plasma exchanges. The fact that the patient entered into remission soon after delivery of a healthy child by cesarean section in the 37th gestational week shows that in this case pregnancy activated an unknown factor which does not cross the placenta and which can be removed by plasmapheresis.     

Extramedullary haemopoiesis after bone marrow transplantation

44 patients underwent bone marrow transplantation (BMT) for treatment of severe aplastic anaemia or haematological malignancies. During their post-transplant phase all patients had erythroblasts and granulocytic precursors in their peripheral blood. 15 patients died between day +6 and +346 after BMT and autopsies were performed. The sections of all 15 patients revealed extramedullary haemopoiesis in the spleen. Extramedullary haemopoiesis in the liver was found only in those patients who died early (between d +6 and d +21 after BMT). Medullary haemopoiesis, normally only occurring in the vertebral body, was also observed in the shaft of the femur. The present data show that after BMT all tissues with a haemopoietic matrix in ontogenesis can be repopulated with haemopoiesis in the early phase of reconstitution, possibly to compensate for the haemopoietic insufficiency after conditioning therapy. The expansion of haemopoiesis in the later period of up to 1 year after BMT, remains to be explained.     

32P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy

Recently, we reported that aristolochic acid (AA) a naturally occurring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the presence of additional AA-DNA adducts by the 32P-post-labelling method not only in the kidneys, but also in a ureter obtained after renal transplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N6-yl)- aristolactam I (dA-AAI), but also the minor adducts, 7-(deoxyguanosin- N2-yl)-aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relative adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of aristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed ingested the natural mixture of AAI and AAII. 32P-post- labelling analyses of further biopsy samples of one patient showed the known adduct pattern of AA exposure not only in the kidney, but also in the ureter, whereas in skin and muscle tissue no adduct spots were detectable. In an attempt to explain the higher level of the dA-AAI adduct compared to the dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contrast to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the kidney of rats. Our data demonstrate that AA forms DNA adducts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA observed in animals could also be responsible for the urothelial cancers observed in two of the CHN patients.      

Rehabilitation of Geriatric Patients with Cervical Spinal Cord Lesions

The intention of this study was to examine the surgical care and the possibilities of rehabilitation in geriatric patients with cervical spinal cord injuries with regard to the postoperative and outpatient course. Thirty-nine patients (aged at least 66 years) with cervical spinal cord injuries were studied over a 6.5-year period (January 1, 1993 to June 30, 1999). Mean age was 75 years. Thirteen patients had a complete sensomotoric lesion of the spinal cord. Seven of them died in hospital during the postoperative period. Only 2 patients are still alive. Three out of the 26 patients with incomplete lesions of the cervical spinal cord were discharged and sent to nursing homes, 22 patients could be discharged into home care. One patient die in hospital. The period of primary rehabilitation was 204 days in patients with complete and 105 days in those with incomplete lesions compared to 85 days in quadriplegic patients overall. In conclusion, a high mortality rate in geriatric patients with complete paraplegia was seen, with only one third of these patients still alive after 1 year. In contrast, 68% of the patients with incomplete lesions survived for more than 1 year, with the majority living in their own houses.     

The impact of long-term participation in the supplemental nutrition assistance program on child obesity

Participation in the Supplemental Nutrition Assistance Program (SNAP) reached an all-time high of 40.2 million persons in March 2010, which means the program affects a substantial fraction of Americans. A significant body of research has emerged suggesting that participation in SNAP increases the probability of being obese for adult women and has little effect on the probability for adult men. However, studies addressing the effects of participation on children have produced mixed results. This paper examines the effect of long-term SNAP participation on the Body Mass Index (BMI) percentile and probability of being overweight or obese for children ages 5-18 using data from the National Longitudinal Survey of Youth 1979 Children and Young Adults data set. An instrumental variables identification strategy that exploits exogenous variation in state-level program parameters, as well as state and federal expansions of the Earned Income Tax Credit (EITC), is used to address the endogeneity between SNAP participation and obesity. SNAP participation is found to significantly reduce BMI percentile and the probability of being overweight or obese for boys and girls ages 5-11 and boys ages 12-18. For girls ages 12-18, SNAP participation appears to have no significant effect on these outcomes.