Expanding the spectrum of SOX1-antibodies in neuropathy: the coexistence of anti-SOX1 and Guillain-Barré syndrome-a case report.

BACKGROUND AND AIMS: Antibodies against SOX1 (or anti-glial nuclear antibody, AGNA) are partially characterized onconeural antibodies, firstly described in association with small cell lung cancer (SCLC). Lambert-Eaton myasthenic syndrome is the most frequent paraneoplastic syndrome (PNS) found in patients with anti-SOX1-antibody positivity. Other associations are chronic axonal polyneuropathy, paraneoplastic limbic encephalitis, and paraneoplastic cerebellar degeneration. METHODS: We describe a case of Guillain-Barré syndrome (GBS) with classical demyelinating phenotype associated with a positivity for anti-SOX1-antibodies. RESULTS: A therapy with intravenous immunoglobulin led to progressive clinical improvement. After 12 months, clinical and neurophysiological pictures showed complete recovery. A thorough paraneoplastic screening was negative for underlying tumors. CONCLUSIONS: This is the first case of GBS associated with anti-SOX1-antibodies described in literature. Although the concept of paraneoplastic GBS is controversial, different cases have been reported and GBS is considered a non-classical paraneoplastic syndrome. Our case expands the anti-SOX1-antibody clinical spectrum with relevant implications for the clinical practice.

Occurrence of septic shock in a patient submitted to emergency cerclage following a negative amniocentesis: report of a case.

BACKGROUND: Second trimester emergency cerclage is an option for pregnant women presenting bulging fetal membranes. Despite a significant prolongation of pregnancy might be achieved, serious fetal and maternal events have been reported. Exclusion of infections through preprocedure amniocentesis has been proposed. METHODS: A 37-year-old woman, gravida 4 para 1, was admitted at 21 weeks of gestation to our University Hospital due to bulging fetal membranes. An amniocentesis was performed in order to exclude an actual amniotic infection. Our Microbiology Department found a negative amniotic culture for bacteria and Mycoplasma and a normal glucose and interleukin-6 level, so a cervical cerclage was performed. The patient was discharged home on oral erythromycin. RESULTS: After 48 h, the patient complained of hyperpyrexia, shivers and reduced fetal movements. Ultrasound at admission showed absent cardiac activity and after cerclage removal a non-viable fetus was delivered vaginally. Piperacillin and tazobactam were started, but the clinical course of the patient deteriorated and she developed a cold septic shock and was submitted to hysterectomy and transferred to the ICU of our hospital. CONCLUSION: This report heralds that even after negative amniocentesis, a life-threatening infection may not be excluded in women candidate for emergency cerclage due to bulging fetal membranes.

New onset status epilepticus in influenza associated encephalopathy: The presenting manifestation of genetic generalized epilepsy.

We hereby present a case of a young woman with no history of seizures or epilepsy who experienced a de novo generalized Non Convulsive Status Epilepticus (NCSE) followed by encephalopathy lasting for several days during influenza B infection. Influenza can have a broad spectrum of presentation ranging from an uncomplicated illness to many serious conditions as is the case of influenza associated encephalitis/encephalopathy (IAE). In this context however, it is possible to observe seizures and/or status epilepticus as the presenting manifestation of a genetic generalized epilepsy.

Uracil salvage pathway in PC12 cells.

The salvage anabolism of uracil to pyrimidine ribonucleosides and ribonucleotides was investigated in PC12 cells. Pyrimidine base phosphoribosyl transferase is absent in PC12 cells. As a consequence any uracil or cytosine salvage must be a 5-phosphoribosyl 1-pyrophosphate-independent process. When PC12 cell extracts were incubated with ribose 1-phosphate, ATP and uracil they can readily catalyze the synthesis of uracil nucleotides, through a salvage pathway in which the ribose moiety of ribose 1-phosphate is transferred to uracil via uridine phosphorylase (acting anabolically), with subsequent uridine phosphorylation. This pathway is similar to that previously described by us in rat liver and brain extracts (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273; Mascia et al., Biochim. Biophys. Acta 1472 (1999) 93). We show using intact PC12 cells that they can readily take up uracil from the external medium. The analysis of intracellular metabolites reveals that uracil taken up is salvaged into uracil nucleotides, with uridine as an intermediate. We propose that the ribose 1-phosphate-dependent uracil salvage shown by our in vitro studies, using tissues or cellular extracts, might also be operative in intact cells. Our results must be taken into consideration for the comprehension of novel chemotherapeutics' influence on pyrimidine neuronal metabolism.

Metabolism of 1,3-cyclohexadiene by isolated rat liver cells.

The metabolism of 1,3-cyclohexadiene by hepatocytes from phenobarbital induced rat has been investigated. Parenchymal cells were obtained by liver perfusion with a hyaluronidase-collagenase mixture. The addition of the diene to a suspension of hepatocytes gave rise to a type I difference spectrum indicating the formation of an enzyme-substrate complex with cytochrome P-450. The subsequent metabolic pathway of 1,3-cyclohexadiene has been shown to involve, as the first step, the formation of 1,2-epoxy-3-cyclohexene, which is rapidly hydrolyzed to trans-3-cyclohexene-1,2-diol and trans-2-cyclohexene-1,4-diol by a non-enzymatic process. The monoepoxide could not be detected in the incubation medium because of its high reactivity. Therefore, kinetic parameters of the epoxidation reaction were determined by following the rate of production of the diols. When incubated with hepatocytes, trans-3-cyclohexene-1,2-diol, the main product of 1,3-cyclohexadiene metabolism, elicited a reverse type I spectrum, indicating that this compound is not a good substrate for the monooxygenase system.

Structure activity relationship of epoxides: different mutagenicity of the two diastereoisomeric 3-bromo-1,2-epoxycyclohexanes.

The mutagenic activities in V79 Chinese hamster cells and the alkylating abilities towards nicotinamide of the two diastereisomeric cis and trans-3-bromo-1,2-epoxycyclohexanes were measured and compared with those of unsubstituted 1,2-epoxycyclohexane and bromocyclohexane. trans-3-Bromo-1,2-epoxycyclohexane exhibited a mutagenic activity 2.5 times higher than that of its cis diastereoisomer, but very similar to that of the parent unbrominated epoxide, whereas the electrophilic reactivities towards nicotinamide were very similar for the three epoxides tested. Bromocyclohexane showed the highest toxicity, but no alkylating ability. The presence of an epoxide hydrolase activity in the V79 Chinese hamster cells used in the mutagenesis tests has been demonstrated using safrole oxide as the substrate, cis-3-Bromo-1,2-epoxycyclohexane, but not its trans diastereoisomer, is hydrolyzed by the enzyme present in microsomal preparations from the V79 cells. The results indicate that for the cycloaliphatic compounds examined: (1) the introduction of a bromide substituent at the carbon adjacent to the oxirane ring does not cause an increase in mutagenicity, (2) the relative stereochemical configuration at the above carbon does affect the biological activity and (3) the significantly different mutagenicity of the two diastereoisomeric 3-bromo-1,2-epoxycyclohexanes is not attributable to a different electrophilic reactivity, but could be related to some specific interaction with detoxifying enzymes present in the V79 Chinese hamster cells used in the biological experiments.

Loss of hepatic monooxygenase activities, glutathione, and 'green pigment' formation after the administration of vinyl-cyclooctane to mice.

Vinylcyclooctane, when administered to mice at 500 mg/kg, produced reduction of microsomal cytochrome P-450, heme, aminopyrine-N-demethylase and ethoxycoumarin-O-deethylase activities with respect to control values; furthermore the hepatic reduced glutathione level was depleted suggesting that glutathione is involved in the vinylcyclooctane metabolism. The reduction of cytochrome P-450 and monooxygenase activities was accompanied by the formation of abnormal 'green pigments'.

Effects of 4-vinylcyclohexene and its main oxirane metabolite on mouse hepatic microsomal enzymes and glutathione levels.

When 4-vinylcyclohexene (VCHE) and 4-vinylcyclohexene monoxide (VCM) were administered to mice at 500 mg/kg, cytochrome P-450, cytochrome b5, NADPH-cytochrome c reductase and aminopyrine-N-demethylase and epoxide hydrolase were induced. Doses of 500 mg/kg of VCHE, VCM and 4-vinylcyclohexene diepoxide depleted rapidly hepatic reduced glutathione levels suggesting that glutathione is probably involved in the metabolism of VCHE.

Metabolism of vinylcyclooctane and partition ratio between epoxide formation and cytochrome P-450 destruction.

Vinylcyclooctane (VCO), which binds to the active site of cytochrome P-450 (P-450) giving a type I difference spectrum, has been found to form the corresponding epoxide as the main metabolite on treatment with liver microsomal monooxygenase obtained from phenobarbital-treated or untreated mice. During this metabolic process about 40% of the microsomal P-450 isozymes are destroyed, but the remainder still demethylates aminopyrine. Approx. 180 molecules of VCO are turned over and 132 of epoxyethylcyclooctane (EECO) are formed for each destructive event.

High sensitivity of Chinese hamster epithelial liver cells to toxic analogues of purines.

The resistance of Chinese hamster epithelial liver cells (CHEL) and Chinese hamster fibroblasts (V79) towards toxic purine analogues has been determined. The liver cells are more sensitive than fibroblasts to 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP). The hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities of extracts of CHEL cells were lower than those of corresponding extracts of V79. The level of 5'-nucleotidase was about 5-fold higher in the epithelial cells. It appears that HGPRT and APRT activities of extracts of liver epithelial cells are masked or reduced by 5'-nucleotidase activity and other inhibitors. The significance of these findings is discussed.

Application of an epithelial liver cell line, metabolically competent, for mutation studies of promutagens.

Recently numerous attempts have been made to reduce the use of vertebrate animals in laboratory experiments to evaluate general and acute toxicity, mutagenesis and teratogenesis of new drugs or chemicals. One common approach is to use established, proliferating cell lines that preserve differentiated functions such as the competence to metabolize xenobiotics. To this end a continuous Chinese hamster epithelial liver cell line (CHEL cells) was established, cultured as used for mutagenesis studies. Structurally different promutagens, such as 7,12-dimethylbenz[a]anthracene (7,12-DMBA), benzo[a]pyrene (B(a)P), aflatoxin B1 (AB1) and cyclophosphamide (CP), were used in order to check and validate the test system. anti-Chrysene-1,2-diol 3,4-epoxide (CDE) and mitomycin C (MMC) were taken as representatives of direct mutagens. The genetic change induced by the mutagens was quantified by measuring mutation frequencies at the HGPRT locus. Several parameters, such as mutant expression time for each chemical, cell density for selection of mutants and enzymatic characterization for HGPRT phenotype, were examined to establish the optimal assay conditions. All promutagens analyzed significantly affected either the cloning efficiency and/or the mutant frequency of CHEL cells after 24 h of exposure. In addition, various enzyme activities involved in the metabolism of the promutagens were determined in CHEL cells, under the experimental conditions of chemical exposure used in the mutagenesis assay. The enzyme activities were compared with those found in uninduced Chinese hamster liver.

Anti-clastogenic activity of two structurally related pterocarpans purified from Bituminaria bituminosa in cultured human lymphocytes.

Plant-derived isoflavones are currently receiving much attention because of their phyto-estrogenic, antioxidant, anti-mutagenic, and anti-tumor activities. In this study we have evaluated the clastogenic and anti-clastogenic activities in human lymphocytes of two structurally related pterocarpans, iso-flavonoid derivatives, termed erybraedin C and bitucarpin A, recently purified from Bituminaria bituminosa and chemically characterized. Mitomycin C (MMC) and the radio-mimetic bleomycin (BL) were used as reference clastogens. The end point studied was micronucleus formation. The results obtained in this study indicate that erybraedin C and bitucarpin A, when assayed alone, do not affect either the mitotic index or the cell-proliferation index of human lymphocytes. Interestingly, both compounds appear to be non-clastogenic in the range of concentrations used. In contrast, both substances seem to affect significantly the clastogenic effects induced by BL and MMC. A 1-h pre-exposition of the cell culture to erybraedin C was necessary to display its anti-clastogenic potential against BL, whereas bitucarpin A was inactive in this respect, with a structure-activity relationship. In contrast, the clastogenic activity of MMC was significantly reduced by both erybraedin C and bitucarpin A, using either a pre-incubation schedule or simultaneous treatment. These results suggest that the protective effects displayed by the two anti-clastogenic compounds against MMC could be due to the induction or inhibition of cellular reductive metabolic enzymes.

3-isobutyl-1-methylxanthine inhibits the mutagenic activity of 7,12-dimethylbenz[a]anthracene in epithelial liver cells in culture.

The mutagenic activity of 7,12-dimethylbenz[a]anthracene in epithelial liver cells (CHEL) in culture was unaffected by the enhancement of intracellular cAMP induced to different extents and with different mechanisms by forskolin and 3-isobutyl-1-methylxanthine. However, the latter compound exerted antimutagenic effects (> 60%), which may be tentatively ascribed to inhibition of the inducible monooxygenase isoform(s) responsible for the specific biotransformation of 7,12-dimethylbenz[a]anthracene to highly mutagenic metabolites in CHEL cells.

A benzo[a]pyrene metabolite-DNA adduct occurring in the activating cell but not in exogenous DNA.

[3H]Benzo[a]pyrene (BP) and salmon sperm DNA were incubated with hepatocytes from 5,6-benzo-flavone-treated rats. The cellular DNA and the exogenously added DNA were separately isolated, hydrolyzed and chromatographed on a Sephadex LH-20 column. The extracellular DNA yielded 3 peaks of radioactivity in the chromatographic eluate. The cellular DNA contained an additional peak suggesting the formation of a DNA adduct from a metabolite that does not leave the cell.

Alkylating properties and genetic activity of 4-vinylcyclohexene metabolites and structurally related epoxides.

The mutagenicity of the epoxides 4-vinyl-1,2-epoxycyclohexane, 4-epoxyethyl-1,2-epoxycyclohexane, 4-epoxyethyl-1,2-dihydroxycyclohexane, 1,2-epoxycyclohexane and styrene oxide was assayed on the TA100 strain of S. typhimurium and V79 Chinese hamster cells. In the latter cell system, both point mutation (6-thioguanine resistance) and chromosomal damage (anaphase bridges and micronuclei) were scored. Genetic effects were related to the alkylating properties of the epoxides. For this purpose, alkylation of 4-(p-nitrobenzyl)pyridine (NBP) and sodium-p-nitrothiophenolate (NTP) was measured and values for the substrate constant (s) were calculated. 4-Epoxyethyl-1,2-epoxycyclohexane, 1,2-epoxycyclohexane and styrene oxide, characterized by the highest reactivity toward NBP and by an s value in the vicinity of 1, were mutagenic in all test systems. 4-Vinyl-1,2-epoxycyclohexane and 4-epoxyethyl-1,2-dihydroxycyclohexane, characterized by lower NBP reactivity and higher s value (1.30-1.38), did not induce reversion in S. typhimurium or 6-thioguanine-resistant mutants in V79 cells, but were as effective as the 3 other compounds in the induction of chromosomal damage.

Induction of sister-chromatid exchanges by procarcinogens in metabolically competent Chinese hamster epithelial liver cells.

An epithelial cell strain has been established from the livers of male Chinese hamsters (CHEL cells). These cells, which proliferate in culture and retain their metabolic enzymatic activities during several subcultures, were used in a sister-chromatid exchange assay to evaluate the effectiveness of polycyclic aromatic hydrocarbons (PAHs), aflatoxin B1 (AFB1) and cyclophosphamide (CP). The results obtained demonstrate that CHEL cells are metabolically competent to activate different classes of procarcinogens into biologically active metabolites. Moreover, they showed a selective capacity to discriminate chemical carcinogens from noncarcinogens. Thus, the CHEL cell system appears to be a promising alternative to the short-term tests that include cell-free rodent liver homogenate to evaluate new promutagens and/or procarcinogens.

Evaluation of the genetic effects induced by vinyl chloride monomer (VCM) under mammalian metabolic activation: studies in vitro and in vivo.

As part of a programme of investigations on the biological effects of the industrial compound vinyl chloride monomer (VCM), the raw material for the production of polyvinyl chloride (PVC), analyses on the genetic effects by this compound have been done by experiments (in vitro) which have taken mammalian metabolism into account. Vinyl chloride in the presence of purified microsomes (sedimented at 105,000 g) obtained from mouse liver was converted into an active metabolite(s) which produced gene mutations in the yeast Schizosaccharomyces pombe (forward mutation) and gene conversions in two loci of a diploid Saccharomyces cerevisiae. Moreover, the compound was active in the host-mediated assay, when mice were treated with an oral dose of 700 mg/kg. The role is discussed of mutagenicity tests for the prediction of both genetic and carcinogenic risks of chemical compounds in industrial use.

Mutagenicity of 3 structurally related epoxides, with defined stereochemical configuration, in Saccharomyces cerevisiae and in V79 Chinese hamster cells.

3 structurally related epoxides, 3,4-epoxycyclohexene, trans-1,2,3,4-diepoxycyclohexane and trans-3,4-epoxycyclohexane-r-1,trans-2-diol (anti isomer) were tested for their ability to induce both point mutation, mitotic gene conversion and recombination in a diploid strain (D7) of the yeast Saccharomyces cerevisiae, with and without a mammalian microsomal activation system, and the formation of 6-thioguanine-resistant mutants in V79 hamster cells. Genetic effects were related to the alkylating properties of the epoxides, as measured by alkylation of 4-(p-nitrobenzyl)pyridine (NBP). Of the 3 epoxides, only 3,4-epoxycyclohexene, characterized by the highest reactivity towards NBP, induced all genetic effects in both test systems. A marginal activity was shown by trans-1,2,3,4-diepoxycyclohexane only in the yeast. The lack of genetic activity of the anti isomer of 3,4-epoxycyclohexane-1,2-diol, in spite of the formal similarity of its functional groups with those present in mutagenic polycyclic arene epoxydiols, was attributed to the dramatic reduction of lipophilicity of the molecule.

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

The type I binding of some aliphatic epoxides to cytochrome P-450 and their inhibition of mono-oxygenase activity.

The Ks values from type I binding of several aliphatic epoxides to microsomes from phenobarbital-or 3-methylcholanthrene-pretreated or untreated mice have been determined. A good correlation between binding affinity and substrate lipophilicity (as log P octanol/water) was observed. Moreover, a striking correlation between delta Emax and log P was found, pointing to a great importance of lipophilicity for the interaction of these compounds with hydrophobic membranes-bound cytochrome P-450. The Ks values of some parental olefins were determined and found to be of the same extent as those of respective epoxides. This suggests that the epoxides can be still good substrates for the mono-oxygenase. The resulting possible hydroxylation would be a further way of detoxication process. This is also supported by the fact that epoxides inhibit the aminopyrine N-demethylase activity.