The German Thorotrast Cohort Study: a review and how to get access to the data.

It is well known that exposures like those from (226)Ra, (224)Ra and Thorotrast(®) injections increase the risk of neoplasia in bone marrow and liver. The thorium-based radioactive contrast agent Thorotrast(®) was introduced in 1929 and applied worldwide until the 1950s, especially in angiography and arteriography. Due to the extremely long half-life of several hundred years and the life-long retention of the thorium dioxide particles in the human body, patients suffer lifetime internal exposure. The health effects from the incorporated thorium were investigated in a few cohort studies with a German study being the largest among them. This retrospective cohort study was set up in 1968 with a follow-up until 2004. The study comprises 2326 Thorotrast patients and 1890 patients of a matched control group. For those being alive at the start of the study in 1968 follow-up was done by clinical examinations on a biannual basis. For the others, causes of death were collected in various ways. Additionally, clinical, radiological and biophysical studies of patients were conducted and large efforts were made to best estimate the radiation doses associated with incorporation of the Thorotrast. The aim of this paper is to describe the cohort, important results and some open questions. The data from the German Thorotrast Study are available to other interested researchers. Information can be found at http://storedb.org .

Chances and shortcomins of adenovirus-mediated ATP7B gene transfer in Wilson disease: proof of principle demonstrated in a pilot study with LEC rats.

BACKGROUND & AIMS: Wilson disease (WD) is an autosomal recessively inherited copper storage disease due to mutations in the ATP7B gene. It results in impaired biliary copper excretion followed by liver injury leading to cirrhosis. In parallel, copper accumulates in other tissues e. g. basal ganglia of the brain inducing motoric disorders. Phenotypical cure of Wilson disease by liver transplantation raised the question whether gene therapy may represent a successful alternative treatment procedure. To examine the principle feasibility of this approach we investigated the effects of gene transfer using an adenoviral vector construct expressing the human ATP7B cDNA in an established rodent model for WD, the Long-Evans Cinnemon rat (LEC). METHODS: Transduction efficiency was assessed by RT-PCR, Western blot and immunofluorescence analysis. The therapeutic effect was estimated by analyzing holoceruloplasmin and its ferroxidase activity in serum, and the copper content of excrements. Changes in copper homeostasis were determined by positron emission tomography (PET). RESULTS: Successful, but temporary gene transfer was clearly detectable on RNA and protein levels. In parallel the temporary therapeutic effect was documented by restoration of serum holoceruloplasmin and of its ferroxidase activity. Additionally the Ad-ATP7B treated LEC rat revealed a higher (64)Cu content in stool. PET was able to visualize differences in (64)Cu distribution between wild type and LEC rats, indicating its principle usefulness as analytical tool. CONCLUSION: The data demonstrate proof of principle of successful gene therapy in an experimental model of WD. As a consequence of successful but only transient therapeutic effect of adenoviral gene transfer we can now focus more efficient and permanent gene transfer strategies.

Antitumoral activity of a sulphur-containing platinum complex with an acidic pH optimum.

UNLABELLED: Platinum complexes are essential tools for cancer treatment despite their toxic side effects. Here we describe a new platinum complex with sulphurs as complexing atoms (thioplatin). PURPOSE: To demonstrate that the antitumoral activity of a new sulphur-containing platinum compound (thioplatin) depends on a slightly acidic pH. METHODS: Platinum uptake by tumour cells and interaction with DNA was determined at slightly acidic or alkaline pH. To demonstrate low in vivo toxicity the effects of thioplatin on body weight, blood urea nitrogen, white blood cell count and the histopathological appearance of small intestines and kidneys were evaluated at doses that displayed antitumoral effects against human small-cell lung cancer and human colorectal cancer xenotransplants in nude mice. RESULTS: The slightly acidic pH optimum of thioplatin was proven by the altered electrophoretic mobility of plasmid DNA, quantitation of the platinum content in the DNA of tumour cells and cytotoxicity studies. Thioplatin displayed antitumoral activity without severe side effects such as weight loss, renal ischaemia, destruction of villi in the small intestine or leukopenia as observed at comparable doses of cisplatin. Furthermore, probably due to its lipophilic nature, thioplatin was taken up readily even by cisplatin-resistant cells. In vivo studies with human tumour xenografts in nude mice showed a therapeutic index of thioplatin five to ten times higher than that of cisplatin.

Protein phosphorylation degree: determination by capillary liquid chromatography and inductively coupled plasma mass spectrometry.

Capillary liquid chromatography (muLC) interfaced to inductively coupled plasma mass spectrometry (ICPMS) is introduced as a new micromethod to determine the phosphorylation degree in phosphoproteins and phosphopeptides containing cysteine and/or methionine residues. The stoichiometric phosphorus to sulfur (31P to 32S) ratio is experimentally determined by muLC-ICPMS and converted into the degree of phosphorylation using protein/ peptide sequence information. The method is applied to the phosphoproteins beta-casein, beta-casein, and recombinant protein kinase A catalytic subunit and to synthetic phosphopeptides. The accurate data obtained by muLC-ICPMS allow quantitative assessment of the compound-specific discrimination of the electrospray ionization process between nonphosphorylated and phosphorylated proteins and peptides.

Analysis of protein phosphorylation by capillary liquid chromatography coupled to element mass spectrometry with 31P detection and to electrospray mass spectrometry.

A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.

Daily but not pulse calcitriol therapy improves growth in experimental uremia.

Calcitriol (C) pulse therapy is widely used to suppress secondary renal hyperparathyroidism. However, high C serum concentrations may have an antiproliferative effect on growth cartilage cells and may suppress growth rate. The study was designed to evaluate whether daily C and pulse C therapy have differential effects on growth in uremic rats. Female Sprague-Dawley rats (150 g, n=5-10 per group) underwent two-stage subtotal nephrectomy (U). The duration of uremia was 14-18 days. The animals were fed a standard diet or a diet with a low-calcium content. Rats on a low-calcium diet were randomized for recombinant human growth hormone (rhGH) treatment (2.5 IU/kg per day) or solvent. C was injected subcutaneous twice daily (15 pmol/day) or intraperitoneal (105 pmol) twice per week. Weight gain and length gain was determined weekly. After sacrifice, total body calcium was determined by total body neutron activation analysis. Bone micromorphometric analysis of third lumbar vertebra and double staining with tetracycline for determination of mineralization rate were performed. Whereas daily C significantly increased total body length gain within 2 weeks, pulse C did not (U solvent 4.0+/-0.3 cm, UC bolus 4.3+/-0.4 cm, UC daily 5.3+/-0.3 cm, P<0.05). A low-calcium diet reduced and rhGH increased basal length gain and weight gain; regardless of these preconditions, daily but not bolus C increased length gain significantly. C both daily and in bolus form reduced bone osteoid content, but daily C improved mineral apposition rate more than C bolus. Total body calcium corrected for body weight decreased with a low-calcium diet, was lowest with concomitant rhGH treatment, and was not improved by C. In conclusion, daily but not bolus C treatment improves growth in uremic rats.

Analysis of radon-associated squamous cell carcinomas of the lung for a p53 gene hotspot mutation.

Squamous cell lung carcinomas (SCC) from former employees of the Wismut uranium mining company (Saxony, Germany) were obtained from the Stollberg Archive in order to screen for p53 tumour suppressor gene codon 249 arg-->met hotspot mutations, a putative molecular bio-dosimeter of alpha-particle (radon) exposure (Taylor et al (1994) Lancet 343: 86-87; McDonald et al (1995) Cancer Epidemiol Biomarkers Prevent 4: 791-793). Of the 29 archived samples of SCC meeting quality criteria for DNA analysis by polymerase chain reaction (PCR) and Haelll restriction enzyme digestion, two tumours were found that harboured this mutation. DNA sequencing confirmed the presence of a G to T base substitution within the Haelll site spanning codons 249 and 250 of the p53 gene that results in replacement of arginine (wild-type) by methionine at residue 249. When these data are combined with those from our previous study of tumours from the Stollberg Archive in which 50 lung tumours were examined, (including nine SCCs), we conclude that the G-->T (arg-->met) codon 249 mutation prevalence in the Wismut miner cohort is not sharply elevated in lung cancers in general (two mutations/79 tumours), or specifically in SCCs of the lung (two mutations/38 SCC) when compared to data from lung cancer patients with no reported occupational exposure to radon gas.

Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species.

Previous studies have shown that a high proportion (5/6) of human liver angiosarcomas (ASL) associated with exposure to vinyl chloride (VC) contains a GC-->AT mutation at the Ki-ras codon 13. This mutation, however, has not been found in 5 ASL or 2 hepatocellular carcinomas (HCC) induced in rats by VC. These 2 HCC did contain a mutation at codon 61 of the Ha-ras gene. In order to extend this study and further explore the mechanisms of tumour induction, an additional 6 ASL and 6 HCC induced in rats by VC were analysed for ras gene point mutations, as well as 10 rat and 10 murine ASL induced by vinyl fluoride (VF), and 5 ASL, 6 Kupffer cell sarcomas, 4 HCC and 2 cholangiocellular carcinomas induced by Thorotrast in rats. Tumour DNA was analysed by PCR-SSCP and direct sequencing. None of the rodent ASL contained a mutation at codon 13 of the Ki-ras gene showing that the ras gene mutational pattern is species-specific. The CAA-->CTA mutation, previously found at codon 61 of the Ha-ras gene in rat HCC, was observed in 5 further VC-induced HCC but was not detected in the Thorotrast-induced HCC, suggesting carcinogen-specificity. This mutation was also absent in VC-induced ASL, which supports the cell-specificity of the ras mutational pattern in chemically induced tumours. No predominant mutation was detected in VF- and Thorotrast-induced tumours. Thus, a given mutation in a tumour may be carcinogen-specific but also depend on the species and the cell type.

German uranium miner study--historical background and available histopathological material.

Mining activities in the former German Democratic Republic were documented as early as 1168 in the ore mountains (Erzgebirge) of Saxony. Silver, bismuth, cobalt, nickel and tungsten were mined from then up to the end of the 19th century. After the Second World War, the Soviet Occupation Authorities reopened the old silver mines in Saxony to mine uranium for the Soviet nuclear industry. About 400, 000 workers produced a total of 220,000 tons of uranium during the years 1946 to 1990. After the reunification of Germany, the archive of the Institute of Pathology of the mining area was opened for research. It contains protocols of 28,975 autopsy cases and about 400,000 slides collected from 1957 to 1992, about 66,000 tissue blocks, and 238 whole lungs. From the autopsy cases, 17,466 could be identified as workers of the uranium mining company. The remainder of the cases were in the population of the mining area. A comparison of the frequencies of malignancies of male workers older than 15 years with those of the population of the mining area for the years 1957 to 1989 demonstrates a significantly higher percentage of lung cancer among the uranium miners. There was no significant difference for other solid cancers and leukemias.

German uranium miner study--pathological and molecular genetic findings. German Uranium Miner Study, Research Group Pathology.

Uranium miners of the former Wismut company in Germany form the largest cohort of workers exposed to (222)Rn and dust in the world. The German Uranium Miner Study, Research Group Pathology, is evaluating the central pathology archive of the Wismut company. The main tasks of our study are pathological-anatomical and molecular genetic investigations of 28,975 autopsy cases and the evaluation of mining pollutants in the lungs by neutron activation analysis. As part of an observer agreement study, lung tumors are classified according to the WHO/IASLC classification and nontumorigenic lung disorders are registered. Lung tumors were analyzed for the presence of a proposed radon-specific mutation in the TP53 gene (formerly known as p53). Interim results are: (a) In the years 1957 to 1965, a high rate (69%) of small cell carcinomas was found which had declined to 34% by 1990. (b) The percentage of the deceased who suffered from silicosis is not higher in the group of lung tumors than in other tumor groups or the nontumor group. (c) The hypothesis of a radon-characteristic hotspot mutation in the TP53 tumor suppressor gene is not supported by our investigations. (d) Neutron activation analysis demonstrates that uranium, arsenic, chromium, cobalt and antimony can be found in tissue samples from the miners even when they had stopped working more than 20 years before death.

The german thorotrast study: recent results and assessment of risks.

The German Thorotrast study comprises 2,326 patients and 1,890 controls. Forty-eight Thorotrast patients and 239 controls are still alive and are invited for a follow-up examination every 2 years. In the deceased patients, the following neoplastic diseases with excess rates were registered (Thorotrast/controls): liver cancer (454/3); cancer of the bile ducts, including gallbladder (42/7); myeloid leukemia (40/7); myelodysplastic syndrome (30/4); plasmacytoma (10/2); non-Hodgkin's lymphoma (15/5); bone sarcoma (4/1); malignant peritoneal or pleural mesothelioma (9/0). Dose calculations are based on results of whole-body counting, X-ray films, and data obtained from the hospital records on the volume of Thorotrast injected. For liver cancer, the cumulative risk estimate was calculated to be 40 per 10(4) person Sv (radiation weighting factor = 20). These figures are close to the results of the Danish study and are comparable to the results of the Life Span Study of A-bomb survivors after 40 years at risk with 18 to 48 liver cancers per 10(4) person Sv. For hematopoietic malignancies, the cumulative risk was calculated to be about 7 per 10(4) person Sv (radiation weighting factor = 20). This risk estimate is lower by a factor of 10 compared to the results of the Life Span Study.

[Thorotrast-induced liver cancer: results of the German thorotrast study]. / Thorotrast-induzierte tumoren der Leber. Ergebnisse der Deutschen Thorotraststudie.

AIMS: The X-ray contrast medium Thorotrast, used worldwide between 1930 to 1950 predominantly for arteriography, consisted of a colloidal solution of thorium dioxide. The radioactive thorium-232 (half-life 1.4x10(10) years) is stored lifelong in the organs of the reticulo-endothelial system after intravascular injection, causing chronic exposure to alpha radiation. The aim of the German Thorotrast study is the assessment of radiation late effects and the calculation of risk estimates. MATERIAL AND METHODS: The German Thorotrast study started in 1968 as a cohort study and comprises 2326 Thorotrast patients and 1890 patients of a matched control group. The Thorotrast patients who were still alive at the beginning of the study were examined by X-ray plain films of the upper abdomen and of the injection site of the contrast medium as well as by whole-body counter measurements. At the beginning we offered the patients ultrasound and later on CT and MRI at regular intervals for early detection of liver cancer. RESULTS: To date 454 primary liver cancers have been registered in the group of Thorotrast patients compared to 3 cases in the control group. With the help of modern imaging methods relatively small liver cancers were detected and could be surgically removed. DISCUSSION: There is a correlation between the mean accumulated dose to the liver and the incidence of liver cancer. The cumulative risk for liver malignancies is about 600 diseases per 10(4) persons whose liver was exposed to 1 Gy. Also the incidence of liver cirrhosis is correlated with the mean accumulated dose to the liver.

Nickel subsulfide is genotoxic in vitro but shows no mutagenic potential in respiratory tract tissues of BigBlue rats and Muta Mouse mice in vivo after inhalation.

Carcinogenic nickel compounds are known to induce promutagenic DNA lesions such as DNA strand breaks and DNA adducts in cultured mammalian cells. In standard mutation assays, in contrast, they were found to be either inactive or weakly active. In our in vitro mutation studies in a lacI transgenic embryonic fibroblast cell line, nickel subsulfide (Ni3S2) increased mutation frequency up to 4. 5-fold. We subsequently applied the comet assay and transgenic rodent mutation assays to investigate the DNA damaging effect and mutagenic potential of nickel subsulfide in target cells of carcinogenesis. A 2-h in vitro treatment of freshly isolated mouse nasal mucosa and lung cells with nickel subsulfide clearly induced DNA fragmentation in a concentration dependent manner. The strong effect was not seen in the same cell types following inhalative treatment of mice and rats, leading only in the mouse nasal mucosa to high DNA damage. When the same inhalative treatment was applied to lacZ and lacI transgenic mice and rats, the spontaneous mutation frequency of these target genes in the respiratory tissues was not increased. These results support a recently proposed non-genotoxic model of nickel carcinogenesis, which acts through gene silencing via DNA methylation and chromatin condensation. This model may also explain our in vitro mutation data in the lacI transgenic cell line, in which nickel subsulfide increased mutation frequency, but in about one-third of the mutants, molecular analysis did not reveal any DNA sequence change in the coding region of the lacI gene despite of the phenotypic loss of its function.

Partition of thorium between organs of monkeys injected with thorotrast: implications for alpha-particle dosimetry.

Risk estimates for alpha-particle-induced malignancies have been based mainly on studies of Thorotrast patients, but certain aspects of its deposition in the body have been at issue: the partition between the liver, spleen and red bone marrow, and the deposition at lower concentrations in other organs, such as muscle and fat, which may contribute to the risk. To supplement the existing data for humans, thorium concentrations were measured in the organs of two female monkeys 3-4 years after injection with Thorotrast. Relative deposits (liver:spleen:red bone marrow) were 54:6:41 and 75:4:21, in better agreement with the most recent observations in Thorotrast patients than with previous reports. Whereas the human testis had ranked among intermediate-level organs such as the adrenal glands and pancreas, the ovary of the monkey was among the organs with the lowest concentrations. The data suggest that risk factors for induction of malignancies by alpha-particle irradiation should be re-examined.

Hepatic failure and liver cell damage in acute Wilson's disease involve CD95 (APO-1/Fas) mediated apoptosis.

Wilson's disease can result in fulminant liver failure due to hepatic copper overload. The CD95 system mediates apoptosis and has been demonstrated to be involved in liver disease. In this study CD95 mediated apoptosis was investigated in patients with fulminant hepatic failure in the course of Wilson's disease and in an in vitro model of copper treated human hepatoma cells. In patients, hepatic expression of CD95 and CD95L mRNA and apoptosis were detected. Copper overload in vitro resulted in hepatocytic apoptosis which could be reduced with a neutralizing anti-CD95L antibody. Copper treatment of hepatocytes results in activation of the CD95 system and induction of apoptosis which is operative during the course of hepatic failure in acute Wilson's disease.

p53 gene mutation analysis in tumors of patients exposed to alpha-particles.

The p53 gene was examined for point mutations in archived, alpha-radiation-associated lung and liver cancers. Lung tumors of 50 uranium miners in Germany were screened by restriction fragment length analysis for the putative hotspot mutation at codon 249 (Arg-->Met) previously detected in a significant fraction of miners from the Colorado Plateau, USA. This mutation has been proposed as a marker of radon exposure. None of the tumors we examined harbored the hotspot mutation. Five of the 50 tumors, however, did indeed harbor exon 7 mutations, as determined by subsequent mutation analysis of exon 7. These mutations were dispersed among various codons and may be attributable to heavy tobacco smoking in this cohort. In support of this interpretation, we found no mutations in exons 5-8 of the p53 gene in 13 iatrogenic liver cancers induced by injection of Thorotrast, an alpha-emitting radiocontrast agent. We propose that if the p53 tumor suppressor gene is a target for the carcinogenic action of alpha-particle radiation, loss of suppressor function may occur preferentially by mechanisms such as intrachromosomal deletions, rather than by base substitution mutations.

Late diagnosis of Wilson's disease in a case without onset of symptoms.

Wilson's disease (WD) was diagnosed on the basis of a liver biopsy, blood investigations and a radio copper test in a 60-year-old, neurologically normal female with uncharacteristic gastrointestinal complaints. Since this patient never developed symptoms indicative for WD this case suggests the possibility of a subclinical course of untreated WD.

Translocation of Thorotrast in the body.

To investigate whether there is a permanent translocation of Thorotrast in the body, the liver of male Lewis rats was removed 4 weeks after injection of Thorotrast (300 microliters) and replaced by a donor liver. In half of the animals the spleen was removed as well. Measuring the 232Th content in the donor liver at different times after implantation demonstrated a permanent transport of 232Th into the surrogate organ. After 231 days a 232Th depot of about 1.1 mg was found, representing about 3% of the total body burden. The additional removal of the spleen resulted in a significantly lower transport of 232Th into the implanted liver. Histological examinations of the donated livers revealed increasing local concentration of Thorotrast granules, leading to the development of conglomerates. A comparable translocation of Thorotrast was verified in two humans who required liver transplantation more than 40 years after Thorotrast injection.

The effects of Thorotrast and quartz on the induction of lung tumors in rats.

In a long-term animal study, the combined and separate effects of Thorotrast (colloidal 232ThO2) and silica dust on the induction of lung tumors were investigated. Female Wistar rats were exposed for 29 d to aerosol concentrations of quartz of either 6 mg m-3, 30 mg m-3, or 0 mg m-3 (6 h d-1, 5 d wk-1). After inhalation, one-half of all exposed animals received a single intravenous injection of enriched Thorotrast (600 microL, 2960 Bq 228 Th mL-1). In all quartz-exposed groups the incidence of benign and malignant lung tumors turned out to be more than 40%. The additional Thorotrast treatment (lifelong exhalation of 220Rn) led to a marked shortening of latency times (first lung tumor was found 1 y after treatment) and to a higher total incidence in the animals exposed to 30 mg m-3 quartz (57 of 87 animals with lung tumors = 65.5%). In the group treated only with Thorotrast, three of 87 animals developed lung tumors. Statistical methods that correct for intercurrent mortality showed a significant increase of the lung tumor risk with respect to Thorotrast treatment, even for the low quartz groups with nearly similar incidences of lung tumors (in the group with ThO2, 39 out of 87 = 44.8%; in the group without ThO2, 37 out of 82 = 45.1%). The tumors were found predominantly in the peripheral regions of the lung and were preceded by proliferation and hyperplasia of the alveolar and bronchiolar epithelium. The results demonstrate a pronounced interactive effect of quartz and Thorotrast on carcinogenesis of the lung. The underlying possible mechanisms are discussed.