Cancer spectrum and frequency among children with Noonan, Costello, and cardio-facio-cutaneous syndromes.

BACKGROUND: Somatic mutations affecting components of the Ras-MAPK pathway are a common feature of cancer, whereas germline Ras pathway mutations cause developmental disorders including Noonan, Costello, and cardio-facio-cutaneous syndromes. These 'RASopathies' also represent cancer-prone syndromes, but the quantitative cancer risks remain unknown. METHODS: We investigated the occurrence of childhood cancer including benign and malignant tumours of the central nervous system in a group of 735 individuals with germline mutations in Ras signalling pathway genes by matching their information with the German Childhood Cancer Registry. RESULTS: We observed 12 cases of cancer in the entire RASopathy cohort vs 1.12 expected (based on German population-based incidence rates). This corresponds to a 10.5-fold increased risk of all childhood cancers combined (standardised incidence ratio (SIR)=10.5, 95% confidence interval=5.4-18.3). The specific cancers included juvenile myelomonocytic leukaemia=4; brain tumour=3; acute lymphoblastic leukaemia=2; rhabdomyosarcoma=2; and neuroblastoma=1. The childhood cancer SIR in Noonan syndrome patients was 8.1, whereas that for Costello syndrome patients was 42.4. CONCLUSIONS: These data comprise the first quantitative evidence documenting that the germline mutations in Ras signalling pathway genes are associated with increased risks of both childhood leukaemia and solid tumours.

A Novel SLC6A8 Mutation in a Large Family with X-Linked Intellectual Disability: Clinical and Proton Magnetic Resonance Spectroscopy Data of Both Hemizygous Males and Heterozygous Females.

X-linked creatine transport (CRTR) deficiency, caused by mutations in the SLC6A8 gene, leads to intellectual disability, speech delay, epilepsy, and autistic behavior in hemizygous males. Additional diagnostic features are depleted brain creatine levels and increased creatine/creatinine ratio (cr/crn) in urine. In heterozygous females the phenotype is highly variable and diagnostic hallmarks might be inconclusive. This survey aims to explore the intrafamilial variability of clinical and brain proton Magnetic Resonance Spectroscopy (MRS) findings in males and females with CRTR deficiency. X-chromosome exome sequencing identified a novel missense mutation in the SLC6A8 gene (p.G351R) in a large family with X-linked intellectual disability. Detailed clinical investigations including neuropsychological assessment, measurement of in vivo brain creatine concentrations using quantitative MRS, and analyses of creatine metabolites in urine were performed in five clinically affected family members including three heterozygous females and one hemizygous male confirming the diagnosis of CRTR deficiency. The severe phenotype of the hemizygous male was accompanied by most distinct aberrations of brain creatine concentrations (-83% in gray and -79% in white matter of age-matched normal controls) and urinary creatine/creatinine ratio. In contrast, the heterozygous females showed varying albeit generally milder phenotypes with less severe brain creatine (-50% to -33% in gray and -45% to none in white matter) and biochemical urine abnormalities. An intrafamilial correlation between female phenotype, brain creatine depletion, and urinary creatine abnormalities was observed. The combination of powerful new technologies like exome-next-generation sequencing with thorough systematic evaluation of patients will further expand the clinical spectrum of neurometabolic diseases.

Diagnostic accuracy of noninvasive polymerase chain reaction testing for the determination of fetal rhesus C, c and E status in early pregnancy.

OBJECTIVE: The aim of the study was to determine the sensitivity, specificity and accuracy of noninvasive tests for the fetal rhesus CcEc (RHCE) alleles C, c and E in early pregnancy. DESIGN: A prospective clinical trial was carried out to evaluate diagnostic accuracy. SETTING: Women were recruited at four centres specialising in prenatal diagnosis. Peripheral blood and amniotic fluid samples were obtained and sent to a single laboratory for analysis. SAMPLE: A total of 233 tests (46 for C, 87 for c and 100 for E) were performed on 181 specimens obtained from pregnant women at weeks 12 to 28 (median week 16) of gestation. METHODS: Following automated extraction of fetal DNA from maternal plasma, two different real-time polymerase chain reaction (PCR) protocols were used for the detection of the C, c and E alleles of RHCE. The results of the PCR were compared with genotyping results for the amniotic fluid. MAIN OUTCOME MEASURES: Failure rate, sensitivity, specificity and accuracy were the main outcome measures. RESULTS: Unequivocal results were obtained for all specimens. With the first PCR protocol, the sensitivity was 100% for C, 38% for c and 59% for E. In contrast, with the second protocol, the sensitivity for C, c and E was 100%. The specificity for all assays was found to be between 99% and 100%. CONCLUSIONS: A highly accurate protocol has been identified for the detection of fetal RHCE alleles in maternal plasma in early pregnancy. This noninvasive approach can be considered as a useful test in the management of pregnancies with anti-c, anti-E or anti-C alloimmunisation.

[Anticoagulation during pregnancy following an artificial heart valve replacement]. / Antikoagulation in der Schwangerschaft nach künstlichem Herzklappenersatz.

HISTORY AND CLINICAL FINDINGS: A 23-year-old woman had received a mechanical bileaflet mitral valve prosthesis because of severe mitral valve insufficiency caused by an acute bacterial endocarditis with vegetations. One year after the operation the patient suffered on two miscarriages under oral anticoagulation by phenprocoumon. Present, she was referred to our center with the question of conversion to low molecular weight heparine because of continued yearning for a baby. INVESTIGATIONS: At admission the woman was in good general and nutritional condition. Echocardiography showed a regular prosthetic function. Blood analysis, electrolyte parameters and enzyme values were normal, further laboratory investigations revealed a factor-V-Leiden-mutation. A chromosomal analysis detected no aberrations. TREATMENT AND COURSE: The oral anticoagulation by phenprocoumon was switched to subcutaneous low molecular weight heparine in therapeutical dosage. Anti-factor-Xa-activity was controlled at regular intervals. Further pregnancy was uneventful for both, mother and child. A healthy infant was born by caesarean section at 40 (th) week of gestation. CONCLUSIONS: Treatment with anticoagulation by phenprocoumon is indispensable for mechanical heart valve protheses. Conversion to low molecular weight heparine is possible in patients who insistent request to conceive. The anticoagulation by low molecular weight heparine avoids teratogenic effects during pregnancy because the placenta is impermeable to that heparin. Furthermore, prophylaxis of thromoses by low molecular weight heparine is probably in almost the same manner as by phenprocoumon.

Oxidative stress in rat cortical neurons and astrocytes induced by paraquat in vitro.

Oxidative stress has been discussed as crucial mechanism of neuronal cell death in the adult brain. However, it was not clear until now whether neurons are more sensitive to oxidative stress than the other cells in the brain, e.g. astrocytes. Therefore both cell types were exposed to oxidative stress provoked by the redox-cycling compound paraquat. Cortical neurons were found to be more sensitive towards paraquat toxicity than astrocytes as shown by MTT and Neutral Red assay, two different cytotoxicity assays. Mitochondrial functions were determined by the mitochondrial membrane potential and intracellular ATP concentrations. Again cortical neurons were more severely impaired (by paraquat than astrocytes). The production of reactive oxygen species after paraquat exposure was much higher in cortical neurons than in astrocytes and correlated with a higher depletion of GSH (intracellular glutathion). Lipid peroxidation could be shown in astrocytes via the breakdown product malondialdehyde (MDA) whereas in cortical neurons 4-hydroxynonenal (4-HNE) was detected as this endpoint. If and how oxidative stress influences the antioxidant defense was determined via changes in the expression of antioxidant enzymes. Paraquat exposure lead to a 2-3 fold increase of catalase, MnSOD and CuZnSOD mRNA expression in astrocytes. In contrast to astrocytes, in cortical neurons catalase and MnSOD mRNA levels were only marginally elevated above 1.5-fold by treatment with paraquat. Expression levels of glutathione peroxidase (GPx) mRNA were the only one that were not changed in both cell types after paraquat exposure. It is concluded that the more marked increase in expression levels of antioxidant enzymes may render astrocytes more resistant to oxidative stress than neuronal cells.

A 74-bp promoter of the Tnp2 gene confers testis- and spermatid-specific expression in transgenic mice.

During the final stages of spermatogenesis, round spermatids undergo several morphological, biochemical, and physiological modifications which result in the formation of mature spermatozoa. One of these is the nuclear condensation, achieved by the replacement of somatic-type and testis-specific histones by transition proteins and protamines leading to cessation of transcription several days before the completion of spermiogenesis. Therefore, a strict temporal and stage-specific gene expression is necessary for the correct differentiation of round spermatids into mature spermatozoa. In this study, the 5' regulatory region of rat Tnp2 gene was investigated by primer extension analysis and transgenic mice study. Primer extension analysis revealed a transcription start site which lies 70 bp upstream of the translation start codon. By transgenic mice studies, we demonstrated that a 147-bp 5' untranslated region corresponding to the region -74 to +73 is sufficient to confer testis- and spermatid-specific expression of rat Tnp2 gene.

Bioassay of mannitol and caprolactam and assessment of response to diethylnitrosamine in heterozygous p53-deficient (+/-) and wild type (+/+) mice.

Alternative bioassays of mannitol (MAN) and caprolactam (CAP) were conducted in transgenic p53-deficient mice. Also, to assess the sensitivity of the transgenic mice to a model DNA-reactive carcinogen, the hepatic effects of diethylnitrosamine (DEN) were compared in the wild type background strain of mouse and in the transgenic derivative. Fifty-one male wild type strain C57BL/6 mice p53 (+/+), 8 weeks old, and 51 heterozygous p53 (+/-) C57BL/6 Tac-[KO] Trp53 N5 mice, 8 weeks old, were allocated to six experimental groups as follows: groups 1 (wild type +/+) and 2 (p53 +/-) served as room controls, groups 3 (+/+) and 4 (+/-) were exposed orally (gavage) to 50 mumol/kg body weight DEN weekly for a total of ten doses during the first 10 weeks of the study, group 5 (+/-) was exposed to 15,000 ppm CAP in the diet for up to 26 weeks, and group 6 (+/-) was exposed to 50,000 ppm MAN in the diet for up to 26 weeks. After 10 weeks, liver from control and DEN-exposed mice was used for O4-ethylthymidine (O4-EtT) DNA adduct analysis by the immunoslot blot method. The cell replicating fraction (RF) in the liver was determined by quantification of the percentage of immunohistochemically stained hepatocytes positive for proliferating cell nuclear antigen. No significant or consistent body or liver weight changes were present in any of the treatment groups. No consistent and pertinent changes in RF values were present in any of the treatment groups. None of the tested substances produced neoplasms of any type in p53 (+/-) mice. DEN induced comparable levels of O4-EtT adducts in the liver in both wild type and p53 +/- genotypes, but no morphologic changes were evident in the livers of either genotype. The lack of response to DEN, in spite of formation of DNA adducts, may reflect the resistance to hepatocarcinogenesis of the background C57BL/6 strain of the transgenic, and calls into question the general sensitivity of this transgenic for detection of carcinogenic effects.

Rat transition nuclear protein 2 regulatory region directs haploid expression of reporter gene in male germ cells of transgenic mice.

During spermiogenesis, the successive replacement of somatic histones by basic proteins, the transition nuclear proteins and protamines, allows normal sperm condensation. Transition nuclear protein 2 (TNP2) is transcribed postmeiotically in round spermatids. In order to determine regulatory flanking sequences responsible for stage specific expression of TNP2 gene, different transgenic mice were generated by microinjection of fertilized eggs. We demonstrate here that 525 bp of 5'- and 920 bp of 3'-flanking sequences of rat TNP2 gene could properly and efficiently direct chloramphenicol acetyltransferase gene expression to the postmeiotic male germ cells of transgenic mice. During male germ cell differentiation the first transgene transcripts were observed in round spermatids and translation started 6 days later in elongating spermatids, which is an evidence for posttranscriptional regulation of transgene expression. In contrast, transgenic mice bearing only the 525 bp 5'-flanking sequences of rat transition protein 2 gene and 3'-flanking sequences of the simian virus 40 large T antigen showed low levels of transgene expression in testis. From these results, it can be concluded that the 525 bp 5'-flanking sequences regulate the cell specific expression and the sequences located in 920 bp 3'-flanking region either enhance the transgene expression in the male germ cells or may have a posttranscriptional role in stabilizing the mRNA in addition to its function in delaying the mRNA translation. Using comparative alignment of 5'-flanking of TNP2 genes from different species, the putative regulatory sequences are identified.

Photochemical mutagenicity of phototoxic and photochemically carcinogenic fluoroquinolones in comparison with the photostable moxifloxacin.

Certain fluoroquinolone (FQ) antibiotics that show clinical phototoxicity and experimental photochemical carcinogenicity have been found to interact with ultraviolet-A (UVA) radiation to produce oxidative DNA damage in cultured cells and isolated DNA. To study the biological consequences of oxidative DNA damage in mammalian cells, the photochemical mutagenicity of two photoactive FQs, lomefloxacin and Bay y3118, was studied in V79 cells in comparison with that of the photostable moxifloxacin. Lomefloxacin and Bay y3118 were photochemically mutagenic to V79 cells with UVA irradiation, increasing the mutation frequency by about eightfold (400 microM, 6000 J/m2) and tenfold (50 microM, 1000 J/m2), respectively, whereas no photochemical mutagenicity was observed with moxifloxacin (400 microM, 9000 J/m2). We suggest that the previously reported ability of lomefloxacin and Bay y3118 to photochemically produce oxidative DNA damage, which is known to be mutagenic, may be the basis for the photochemical mutagenicity and the reported photochemical carcinogenicity. The photostable moxifloxacin appears to lack such properties.

Rat cortical neuron cultures: an in vitro model for differentiating mechanisms of chemically induced neurotoxicity.

Various structurally unrelated chemicals [2,5 hexandione, acrylamide, organophosphates like mipafox, beta,beta iminodipropionnitrile (IDPN), 3-nitropropionic acid (3-NP), potassium cyanide (KCN), paraquat, and NMDA (N-methyl-D-apartic acid)] are known to cause degenerative damage of the peripheral or central nervous system. Differentiated neuronal cell cultures obtained from fetal rats have been used to differentiate the mechanisms underlying this type of neurotoxicity. Cytotoxicity as measured by a viability assay was not sensitive enough and had to be supplemented by further endpoints covering effects on cytoskeleton and on the energy state of the cells [glucose consumption, mitochondrial membrane potential and adenosine 5'-triphosphate (ATP) concentration]. Compounds like the delayed neurotoxic organophosphates, exert a selective direct effect on cytoskeleton elements in this model at concentrations distinctly below cytotoxic concentrations. Other compounds, like KCN, paraquat, and 3-NP selectively disrupt the balance between energy supply and demand of the neurons either by interacting with mitochondrial respiration or glycolysis. For these compounds cytoskeletal damage seemed to be secondary to the energy depletion. For NMDA, 2,5 hexandione and acrylamide, both mechanisms may contribute to the neuronal damage. In conclusion, primary cortical neuronal cultures of the rat are well suited to detect a neurotoxic potential and to differentiate its underlying mechanisms. Damage of the cytoskeleton may be considered as an endpoint mechanistically related to degenerative neuropathic effects.

Evidence for translational repression of the SOCS-1 major open reading frame by an upstream open reading frame.

The suppressor of cytokine signalling 1 protein (SOCS-1) belongs to a novel family of cytokine inducible factors which function as inhibitors of the JAK/STAT pathway. While SOCS-1 previously has been described as a single-exon gene, here we present evidence for an additional 5' exon, separated by a 509 bp intron from exon 2. Exon 1 and part of exon 2 contain an open reading frame of 115 nt, ending one nucleotide upstream of the major reading frame. Using SOCS-1-promoter/luciferase constructs, we investigated which sequences are involved in the regulation of SOCS-1 expression. Serial promoter deletion clones indicate the localization and functionality of SP1, interferon-stimulated responsive elements (ISRE), and interferon-gamma-activated sites (GAS) promoter elements in the SOCS-1 5' flanking region. We present evidence that the upstream open reading frame (uORF) represses the translation of the downstream major open reading frame (mORF). Mutating the start codon of the uORF relieves this repression. Our data indicate that expression of the SOCS-1 protein is repressed on translational level by a mechanism, which bears similarities to that postulated for genes like retinoic acid receptor beta2 (RARbeta2), S-adenosylmethionine-decarboxylase (AdoMetDC), Bcl-2, and others.

Nonlinearity of nuclear enlargement in hepatocytes induced by the carcinogen N'-nitrosomorpholine in Ovo.

In this study we investigated the effects of different doses of the carcinogenic nitrosamine N'-nitrosomorpholine (NNM) on the occurrence of enlarged nuclei in embryonic turkey liver in order to evaluate whether this parameter might represent a quantitative indicator of chemically induced hepatocarcinogenesis. Therefore fertile embryo turkey eggs were injected with NNM over a dose range of 125 microg-8 mg/egg at the first day of incubation. After incubation for 24 days, the embryonic livers were removed and processed for histologic evaluation. The induction of hepatocytes with enlarged nuclei (nuclear profiles > 35 microm2 was quantitated morphometrically in hematoxylin and eosin (H&E)-stained sections. The NNM treatment increased both the number of enlarged hepatocyte nuclei and the areas of the individual profiles of the enlarged nuclei in a dose-dependent manner. Exposure to 500 microg-8 mg NNM/egg resulted in a statistically significant increase in the number of hepatocytes with enlarged nuclei. The lower doses of 250 microg and 125 microg NNM/egg showed a similar albeit not significant trend. Signs for cytotoxic effects on the hepatocytes, such as necrosis or enhanced cytoplasmic vacuolization, were observed in tissue samples of embryos exposed to 4 or 8 mg NNM, but not after treatment with lower doses. The dose-effect curve for the induction of the nuclear enlargement was nonlinear, with a moderate slope for lower dose levels of 125-500 microg/egg and a steep slope for higher dose levels of 1-8 mg. Findings in rodents indicate a pathogenic link between the occurrence of enlarged nuclei and hepatocarcinogenesis. Based on the results with NNM, it is suggested that the in ovo model may represent a rapid, convenient, and inexpensive experimental approach for dose effect investigations on chemically induced hepatocarcinogenesis.

Different mechanisms for the photoinduced production of oxidative DNA damage by fluoroquinolones differing in photostability.

Several fluoroquinolone antibacterial agents exhibit an adverse phototoxic effect in humans and are photo-cocarcinogenic in mice. The UV-induced production of reactive oxygen species plays a role in the toxicity and may be involved in carcinogenicity. Four fluoroquinolones were examined for the ability to photochemically produce oxidative damage in naked DNA. The major structural difference in the fluoroquinolones that would have an effect on their photostability is the functionality at the 8-position. At this position, 1-cyclopropyl-7-(2,8-diazbicyclo[4.3.0]non-8-yl)-6, 8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (BAY y3118) contains a chlorine atom, lomefloxacin a fluorine atom, ciprofloxacin a proton, and moxifloxacin a methoxy group. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in calf thymus DNA was assessed by HPLC with electrochemical detection, and strand breaks were measured in pBR322 with agarose gel electrophoresis. The relative photolability of the fluoroquinolones correlated to the extent of production of 8-oxodGuo and strand breaks, with both UVA and UVB irradiation, in the following order: BAY y3118 approximately lomefloxacin > ciprofloxacin > moxifloxacin. Experiments were performed to determine whether the mechanism of damage was due to a type I (radical) or type II (singlet oxygen) pathway. Nitrogen depletion of oxygen resulted in a decrease in the extent of formation of 8-oxodGuo, suggesting that oxygen was involved. The use of selective radical or singlet oxygen inhibitors was inconclusive with respect to which pathway was involved. The use of D(2)O as a solvent, which would extend the lifetime of singlet oxygen, suggested that this species is involved in the formation of 8-oxodGuo by moxifloxacin and ciprofloxacin, but not by lomefloxacin and BAY y3118. Similarly, it was found that singlet oxygen was not involved in strand break formation. Thus, the evidence suggests that fluoroquinolones can photochemically produce DNA damage by both type I and type II mechanisms.

DNA analysis of Huntington's disease: five years of experience in Germany, Austria, and Switzerland.

OBJECTIVE: To review the direct DNA testing for Huntington's disease (HD) in Germany, Switzerland, and Austria from 1993 to 1997, and to analyze the population with regard to age structure, gender, and family history. METHODS: Twelve laboratories (nine in Germany, two in Austria, and one in Switzerland) recorded data pertaining to repeat number, gender, age at molecular diagnosis, and family history of probands. The molecular test was categorized as either diagnostic (for symptomatic individuals), presymptomatic (for individuals at risk), and prenatal (for pregnancies at risk). RESULTS: A total of 3,090 HD patients, 992 individuals at risk, and 24 fetuses were investigated using DNA analysis. The clinical diagnosis was confirmed in 65.6% of patients. A total of 38.5% of individuals at risk inherited an expanded CAG repeat. The female-to-male ratio showed a distinct predominance of women both in the diagnostic and presymptomatic groups. Of the fetuses tested, six were carriers of an expanded CAG repeat. Two pregnancies were interrupted; one pregnancy was not. No information about the parents' decision was obtained for the remaining three pregnancies. CONCLUSIONS: Approximately 20% of the estimated 10,000 HD patients living in Germany, Switzerland, and Austria have been identified by DNA analysis (total population, approximately 100 million; incidence of HD, 1:10,000). Assuming a ratio of HD patients to individuals at risk of 1:3, approximately 30,000 individuals are, in principle, eligible for a presymptomatic test. Less than 3 to 4% of individuals at risk have requested a presymptomatic test. This shows that the assumed enormous request of predictive testing has not occurred. More surprisingly, prenatal diagnoses were found to be rare.

Urinary antigens as markers of papillary toxicity. II: Application of monoclonal antibodies for the determination of papillary antigens in rat urine.

We have previously reported the preparation of monoclonal antibodies specific for antigens localized in the rat renal papilla. Three of the monoclonal antibodies reacting with antigens localized in papillary and cortical collecting duct epithelia were selected for the development of enzyme-linked immunosorbent assay (ELISA)-type assays. The papillary antigens ('PapA') determined in these tests were designated PapAl (applying the monoclonal antibody PapX 5C10), PapA2 (applying the monoclonal antibody PapX 12F6), and PapA3 (applying the monoclonal antibody PapXI 3C7). Using these assays antigen excretion was determined in the urine of rats. Depending on the test compound used. the application route, and the dose, the observed antigen release patterns differed. Whereas after a single intraperitoneal application of 2-bromoethanamine or of propyleneimine an increased release of PapA1 but not of the two other antigens was observed oral application of bromoethanamine had minor effects. In contrast, both a single intraperitoneal application or repeated oral applications of indomethacin resulted in an increased release of all the three antigens. Daily application of ipsapirone in the diet or in drinking water resulted in significantly elevated urinary release of PapAl which increased incrementally for the duration of the application. Release of PapA2 and PapA3 was not affected and remained in the normal range. These results show that with the tests developed changes in the rat renal papilla caused by xenobiotics can be detected early by urinary analysis and monitored during follow-up studies. Moreover. the different antigen release patterns obtained after application of the different compounds suggest a possible differing mode of action.

Preclinical safety evaluation of moxifloxacin, a novel fluoroquinolone.

The toxicity of moxifloxacin (BAY 12-8039), a novel fluoroquinolone with a broad spectrum of antibacterial activity, was evaluated in a comprehensive programme of toxicological studies that included single and multiple dose toxicity studies in rats, mice, dogs and monkeys, reproductive system toxicity studies in rats and monkeys and in-vitro and in-vivo mutagenicity assays. Although moxifloxacin is not intended for long-term clinical use, an accelerated bioassay in target organs to assess carcinogenesis was performed in rats. In addition to the routine toxicological programme required for the development of a drug intended for short-term administration, a major part of the preclinical programme for moxifloxacin comprised studies designed specifically to address the safety issues known to be features of fluoroquinolones, i.e. adverse effects on the central nervous and cardiovascular systems, phototoxicity, arthrotoxicity and oculotoxicity. The results of the toxicological investigations confirmed that the safety profile of moxifloxacin is comparable to those of other fluoroquinolones.

Damage to mitochondrial DNA induced by the quinolone Bay y 3118 in embryonic turkey liver.

Quinolones are a class of antibiotics that induce damage to and loss of DNA from bacteria. The structural organization of bacterial DNA is more similar to eukaryotic mitochondrial DNA (mtDNA) than to eukaryotic chromosomal or nuclear DNA (nDNA). Antibiotics affecting the bacterial genome may therefore preferentially damage mtDNA rather than nDNA. We investigated the effect of a quinolone on mtDNA in avian embryonic hepatocytes in ovo. The quinolone Bay y 3118 (1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl) 6-fluoro-8-chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, chemical structure see Bremm et al. [K.D. Bremm, U. Petersen, K.G. Metzger, R. Endermann, In vitro evaluation of Bay-y 3118, a new full-spectrum fluoroquinolone, Chemotherapy 38 (1992) 376-387] was injected into fertilized turkey eggs 8 days before hatching at doses of 1, 3, 10 and 30 mg per egg. The embryos were removed from the eggs after 4 days and liver samples were shock frozen. Mitochondrial DNA was purified from samples of the embryonic liver. The integrity of mtDNA was investigated by electrophoresis on agarose gels with native mtDNA and with ribonuclease-treated mtDNA. Fluorescent staining of the electrophoresis gels allows the densitometric quantification of the mtDNA of the regular band at 16 kilobases (kb) and the amount of DNA fragments of irregular size (smear). The genotoxic nitrosamine nitrosodiethylamine (NDEA) has previously been shown to reduce the content of mtDNA of the regular size of 16 kb and to induce the occurrence of smaller fragments of mtDNA [H. Enzmann, C. Kühlem, E. Löser, P. Bannasch, Damage to mitochondrial DNA induced by the hepatocarcinogen, diethylnitrosamine in ovo, Mutation Res. 329 (1995) 113-120]. After exposure to 10 and 30 mg Bay y 3118, a dose-dependent induction of damage to the mtDNA was found, whereas exposure to 3 and 1 mg showed no effect. NDEA (25 mg) was used as positive control. Testing chemical compounds in the in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA.

Preclinical safety evaluation of cerivastatin, a novel HMG-CoA reductase inhibitor.

Cerivastatin is a new but structurally distinct 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor ("statin"). It effectively decreases low-density lipoprotein (LDL) cholesterol at 1% of the doses of other currently available statins. The toxicology of cerivastatin was evaluated in a comprehensive program of studies including: (1) single- and multiple-dose toxicity studies in rats, mice, minipigs, dogs, and monkeys; (2) reproductive toxicity studies in rats and rabbits; (3) in vitro and in vivo mutagenicity assays in rats and mice; and (4) carcinogenicity studies in rats and mice. In addition, studies were undertaken to investigate the effects of cerivastatin on lens opacity, testicular tissue, and hemorrhage in dogs. Oral administration of single and multiple doses of cerivastatin over periods ranging from 4 weeks to 24 months was generally well tolerated. Adverse effects were similar to those observed with other statins and primarily involved the liver and muscle tissue. At the high doses used in the toxicologic studies, cerivastatin caused elevations in serum transaminases and creatine phosphokinase levels as well as some degeneration of muscle fibers in rats, mice, dogs, and minipigs. In dogs, the species most sensitive to statins, cerivastatin caused erosions and hemorrhages in the gastrointestinal tract, bleeding in the brain stem with fibroid degeneration of vessel walls in the choroid plexus, and lens opacity. Apart from minor morphologic changes in the testicular tissue of dogs--the only organ for which a comparably low margin of safety was observed--cerivastatin had no significant effects on the male or female reproductive system. Cerivastatin also caused no primary embryotoxic or teratogenic effects. With the exception of cerivastatin-induced effects on the eyes and testicles, administration of mevalonic acid reversed the toxicologic effects of cerivastatin, indicating that the toxic effects were related to its mode of action and not to any intrinsic toxicity of the molecule itself. There was no evidence that cerivastatin had any mutagenic effects and, in contrast to other statins, high doses of cerivastatin did not induce tumors in rats. The main metabolite of cerivastatin was well tolerated systemically in all animals, including dogs. Overall, cerivastatin has a similar toxicologic profile to other statins and is a well-tolerated HMG-CoA reductase inhibitor.