Biomarkers of exposure and potential harm in adult smokers of 3-7 mg tar yield (Federal Trade Commission) cigarettes and in adult non-smokers.

The paper reports levels of 24-h urine nicotine and five of its major metabolites (expressed as nicotine-equivalents) and blood carboxyhaemoglobin as biomarkers of exposure to particulate- and gas-phase cigarette smoke, respectively, from an exploratory pilot study of adult smokers of 3.0-6.9 mg tar delivery (Federal Trade Commission (FTC) method) cigarettes. On multiple occasions over 6 weeks, blood high-sensitivity C-reactive protein (hs-CRP), fibrinogen, HDL- and LDL-cholesterol, and 24-h urine 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) and 11-dehydro-thromboxane B2 (11-dehydro-TxB2) were also evaluated as biomarkers of potential harm. All the biomarkers examined, except for LDL-cholesterol, discriminated with high sensitivity and specificity between adult smokers and non-smokers overall. Except for HDL-cholesterol, all biomarker medians were greater in adult smokers than in non-smokers: urine nicotine-equivalents 64.514 versus < 0.034 nmol mg-1 creatinine (p<0.001), carboxyhaemoglobin 4.0 versus 0.4% saturation (p<0.001), hs-CRP 0.27 versus 0.12 mg dl-1 (p=0.05), fibrinogen 292 versus 248 mg dl-1 (p<0.001), HDL-cholesterol 46 versus 53 mg dl-1 (p=0.003), LDL-cholesterol 119 versus 109 mg dl-1 (p=0.18), urine 8-epi-PGF2alpha 1935 versus 1034 pg mg-1 creatinine (p<0.001) and urine 11-dehydro-TxB2 973 versus 710 pg mg-1 creatinine (p<0.001). All the biomarkers of exposure and most of the biomarkers of potential harm showed no time of sampling (by visit week) effect.

Comparison of fresh and room-aged cigarette sidestream smoke in a subchronic inhalation study on rats.

Two experimental types of cigarette sidestream smoke (SS) were compared in a subchronic inhalation study on rats. Fresh SS (FSS) was generated continuously from the reference cigarette 2R1. Room-aged SS (RASS) was generated by aging FSS for 1.5 h in a room with noninert surfaces with materials typically found in residences or offices. Male Sprague-Dawley rats were head-only exposed to three dose levels of each SS type and to filtered, conditioned fresh air (sham-exposure) for 6 h/day, 7 days/week, for 90 days. Room-aging resulted in decreased concentrations of various SS components, e.g., total particulate matter (TPM) and nicotine, while other components, such as carbon monoxide (CO), were not affected. The CO concentrations were 6, 13, and 28 ppm for both SS types. TPM concentrations were between 0.6 and 8.7 micrograms/liter and thus up to 100-fold above the maximum of average concentrations of respiratory suspended particles reported for environmental tobacco smoke. Slight reserve cell hyperplasia in the anterior part of the nose as well as hyperplastic and metaplastic epithelial changes in the larynx were the only observed dose-dependent findings. The metabolism of benzo(a)-pyrene--as a proxy for polycyclic aromatic hydrocarbon metabolism--was induced in the nasal respiratory epithelium and in the lungs while no effect was seen in the nasal olfactory epithelium. The lowest-observed effect level was 6 ppm CO or 0.6 microgram TPM/liter. Most of the effects seen were less expressed in RASS-than in FSS-exposed rats when compared on the basis of the CO concentrations. When compared on the basis of TPM, these effects were equally pronounced for both SS types, suggesting a major role of particulate matter-associated compounds. All findings reverted to sham control levels following a 42-day postinhalation period.

Metabolism of S-nicotine in noninduced and aroclor-induced rats.

The urinary excretion of nicotine and its metabolites in noninduced and Aroclor-induced male and female rats has been determined following intravenous administration of 2'-[14C]-labeled S-nicotine at a dose of 4.6 mumol/kg. Complete recovery of the administered radioactivity was achieved: 95% in urine and 4% in feces over 96 h and 1% remaining in the body. More than 40 nicotine metabolites were found by radio-HPLC; 19 were identified including the cis/trans-diastereomers of nicotine-N'-oxide and 3'-hydroxycotinine. The urinary metabolite profile and excretion kinetics of nicotine and its metabolites were significantly different between noninduced and Aroclor-induced rats. The major urinary nicotine metabolite in the noninduced rat was cis-nicotine-N'-oxide. In the Aroclor-induced rat, cotinine metabolites were the major metabolites found. Sex differences were found for the urinary nicotine metabolite profile, mainly expressed in the excretion of cis-nicotine-N'-oxide, 29% in the male and 17% in the female noninduced rat, and the excretion of cotinine, 5% in the male and 12% in the female noninduced rat. High stereoselectivity was found for the formation of the cis/trans-diastereomers of nicotine-N'-oxide as well as of 3'-hydroxycotinine, the stereoselectivity being more pronounced in male rats.

DNA-DNA dot hybridization technique used as DNA determination method in the alkaline elution analysis of DNA damage.

A DNA-DNA ('Southern') dot hybridization technique was adapted for use as a quantitative DNA detection method during alkaline elution analysis of irradiated rat cell material. In comparison to standard microfluorometric methods, similar gamma-ray dose-response relationships were obtained with less than 1% of the cell material when the dot hybridization assay was used. When a highly repetitive, long interspersed DNA element of the rat genome is used as a hybridization probe, as few as 10(4) cells of rat tissue or rat cell culture cells per sample with approx. 50 ng of DNA were sufficient to detect single-strand breaks and protein cross-links in the DNA of rat hepatocytes and cells of the nasal epithelium after in vitro gamma-irradiation. Since highly repetitive DNA elements are available from nearly all higher eukaryotes, this alternative approach of detecting DNA in alkaline elution analysis is generally proposed for tissues which yield only low amounts of cell material and/or which are difficult to label by radioactive DNA precursors.

Chromosomal abnormalities and sister-chromatid exchange in bone marrow cells of mice and Chinese hamsters after inhalation and intraperitoneal administration. II. Cyclophosphamide.

The genotoxic effects of cyclophosphamide (CPP), a human and animal carcinogen requiring metabolic activation, were studied in bone marrow cells of mice and Chinese hamsters, analyzing chromosome abnormalities (CA) and sister-chromatid exchange (SCE) after a 2-h inhalation or a single intraperitoneal administration. In order to compare the genotoxicity after the different routes of administration in the dose range of 10-110 mg CPP/kg body weight, the systemic dose obtained by inhalation was calculated from blood concentrations and the inhalation duration after an analysis of the CPP blood kinetics. In NMRI mice the frequency of bone marrow cells with chromosome abnormalities was higher after aerosol exposure than after intraperitoneal administration of comparable CPP doses. In Chinese hamsters the CA frequency was similar with both exposure routes. Inhaled CPP was found to induce a higher frequency of CA and SCE in the bone marrow cells of mice compared to those of Chinese hamsters. The findings suggest that for genotoxins requiring metabolic activation species differences exist with respect to the influence of the route of entry and the sensitivity of bone marrow cells.

Cotinine determination by immunoassays may be influenced by other nicotine metabolites.

Polyclonal rabbit anticotinine antiserum, which can be used for biomonitoring nicotine uptake by the determination of cotinine in body fluids, was checked by a competitive ELISA for its cross-reactivity with nine nicotine metabolites. The highest percentage of relative cross-reactivity (about 30%) was observed with trans-3'-hydroxycotinine, a metabolite which is known to be excreted in 3-fold higher amounts than cotinine in the urine of human smokers. Therefore, it is possible that cotinine determinations performed by immunochemical methods--especially in urine--may yield overestimated cotinine concentrations.

Chromosomal abnormalities and sister-chromatid exchange in bone marrow cells of mice and Chinese hamsters after inhalation and intraperitoneal administration: I. Diepoxybutane.

Diepoxybutane (DEB), a direct-acting animal carcinogen, was found to increase the frequency of structural chromosomal abnormalities (CA) and sister-chromatid exchange (SCE) in bone marrow cells of mice and Chinese hamsters, when inhaled from an aerosol during a 2-h head-only exposure or administered as a single intraperitoneal injection. For the purpose of comparing the genotoxicity in the 2 species, both after inhalation and intraperitoneal administration, the systemic DEB dose obtained by inhalation was determined on the basis of blood concentrations and inhalation duration after the investigation of the blood kinetics. The bone marrow cells of male and female NMRI mice were found to be more sensitive than those of Chinese hamsters to the genotoxic activity of DEB.

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by regulation of hyphal development : An analysis based on scanning electron and light microscopic observations.

The morphological effects of biotin and L-arginine on fruiting body formation of the ascomycete Sordaria macrospora are investigated by scanning electron and light microscopy. Biotin is recognized as an elongation factor and arginine as a branching factor in vegetative and reproductive hyphae. In the absence of exogenous biotin, development is blocked after the ascogonium-core hypha stage of protoperithecial morphogenesis, whereas linear growth of the myceliar front is maintained. The addition of exogenous arginine to a biotin deficient culture induces the formation of numerous side branches even in the older mycelium. Fruiting body formation, however, remains blocked at the protoperithecial stage as before, because of the inability of the side branches to elongate. When biotin and arginine are administered simultaneously, a most vigorous branching and growth are induced in the older mycelium, accompanied by a rapid and maximal formation of fruiting bodies. The results are summarized in a model of the exogenous control of hyphal morphogenesis. The model is designed to explain the relationship between fruiting and hyphal density as well as the edge effect on fruiting body formation.

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes.

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [(14)C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons : I. Molecular properties of the purified isoenzymes.

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD(+) oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: τ1/2 at 40°C=3.0 min; mMDH: stable at 40°C; τ1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3-9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons : II. Kinetic properties of the purified isoenzymes.

Kinetic parameters of the glyoxysomal and mitochondrial malate dehydrogenase (EC 1.1.1.37) of watermelon (Citrullus vulgaris Schrad.) cotyledons were compared. The data were obtained by initial rate experiments at pH 8.5 in both directions of the reaction using homogeneous enzyme preparations. Substrate inhibition at physiologically significant concentrations was observed with reduced nicotinamide adenine dinucleotide (NADH) (50% inhibition at 0.65 mmol·l(-1) NADH), but not with oxaloacetate, L-malate or oxidized nicotinamide adenine dinucleotide. The inhibition of both isoenzymes by 5'adenosine monophosphate was studied. Inhibition was found to be competitive with respect to NADH and non-competitive with respect to oxaloacetate. The apparent inhibitor constants at 200 µmol·l(-1) of the fixed substrates were 3.2 and 1.6 mmol·1(-1) for NADH, and 3.2 and 5.2 mmol·l(-1) for oxaloacetate with the glyoxysomal and mitochondrial isoenzymes, respectively. The energy of activation was determined for oxaloacetate reduction by glyoxysomal (E a =3.14×10(4)J×mol(-1)) and mitochondrial (E a =4.10×10(4) J x mol(-1)) MDH from Arrhenius plots, which exhibited constant slopes throughout the range of thermal stability.Despite considerable structural differences, the results indicate very similar kinetic behaviour of the glyoxysomal and mitochondrial isoenzymes. The physiological significance of the data are discussed in relation to the gluconeogenic processes occuring in cotyledons during germination.

Separation of malate dehydrogenase isoenzymes by affinity chromatography on 5'-AMP-Sepharose.

The mitochondrial and glyoxysomal isoenzymes of malate dehydrogenase (EC 1.1.1.27) from watermelon cotyledons and the mitochondrial isoenzyme from pig heart adsorbed reversibly to 5'-AMP-Sepharose. They were specifically eluted with low concentrations of NADH rather than by NAD. In contrast, the cytoplasmic isoenzymes showed no affinity to the matrix-bound ligand. These binding properties are discussed in terms of structural and regulatory differences of the particulate and soluble malate dehydrogenase isoenzymes. Affinity chromatography on 5'-AMP-Sepharose significantly improved the purification of the particulate malate dehydrogenase isoenzymes with respect to homogeneity, yield, and the number of purification steps. In the case of the glyoxysomal isoenzyme it was the essential procedure to obtain complete purification of the enzyme.

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination.

Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1(-1), accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.

[Purification and characterization of chloroplast carbonate dehydratase from leaves of Lactuca sativa (author's transl)]. / Reinigung und Charakterisierung von Chloroplasten-Carbonat-Dehydratase (Isoenzym I) aus Blättern von Lactuca sativa

Two isoenzymes of carbonate dehydratase were identified in green leaf tissue of Lactuca sativa. Their molecular weights were found to be 195 000 and 250 000. The lighter isoenzyme (I) was further characterized. It is localized in the chloroplast fraction. With polyacrylamide gel electrophoresis in the presence of dodecyl sulphate, subunits with a molecular weight of 34 000 and higher aggregates of this size could be detected. This is interpreted as an indication of an hexameric enzyme structure. The 900-fold purified polymer contained 5 - 6 atoms of zinc. Amino acid composition and inhibition by acetazolamide (Diamox), cyanide, nitrate, azide and ferricyanide are described.