Microsomal ring-fission of cis- and trans-acenaphthene-1,2-diol

1. The ring-fission of cis- and trans-acenaphthene-1,2-diol by rat liver microsomes was studied. 2. 1,8-Naphthalic acid was detected and isolated after microsomal incubations of the diols. 3. The accompanying reduction of NAD(+) was followed spectrophotometrically. 4. The optimum pH for the microsomal reaction was 9.4 for the oxidation of the cis-diol and 9.8 for that of the trans-diol. 5. p-Chloromercuribenzoate and 2,4-dichlorophenol inhibited the reaction. 6. Possible mechanisms for the microsomal ring-fission, involving 1,8-naphthalic aldehyde, are discussed.     

Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO₂ by the tricarboxylic acid cyle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.     

Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO2 by the tricarboxylic acid cycle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.     

Biochemical studies of toxic agents. Metabolic ring-fission of cis- and trans-acenaphthene-1,2-diol

1. The metabolism of cis- and trans-acenaphthene-1,2-diol has been studied after the administration of these compounds to rats by subcutaneous injection and by stomach tube. 2. 1,8-Naphthalic acid has been isolated as its anhydride from the urine of the dosed animals. 3. A spectrophotometric method for the determination of free and conjugated 1,8-naphthalic acid in urine has been developed and has been used in the study of the metabolism of the acenaphthene-1,2-diols. 4. The urine of rats dosed with cis-acenaphthene-1,2-diol by subcutaneous injection was shown by paper chromatography to contain both cis- and trans-acenaphthene-1,2-diol. Similar findings were obtained after the subcutaneous injection of trans-acenaphthene-1,2-diol.     

The metabolism of cis- and trans-decalin

1. The metabolism of cis- and trans-decalin in the rabbit has been investigated. 2. Both hydrocarbons were oxidized to racemic secondary alcohols and excreted as ether glucuronides in amounts equal to about 60% of the dose administered. The principal glucuronides were isolated as triacetyl methyl esters and as sodium salts. 3. cis-Decalin gave rise mainly to (+/-)-cis-cis-2-decalol, together with a little cis-trans-2-decalol, and trans-decalin mainly to (+/-)-trans-cis-2-decalol and a small amount of trans-trans-2-decalol. 4. These results suggest that biological oxidation of the decalins does not occur via a free-radical mechanism. An attempt is made to explain why racemic alcohols are obtained, rather than the more typical optically active products of enzymic reaction, and a mechanism is proposed. It is suggested that enzymes similar to steroid hydroxylases are involved.     

The importance of 3,4-epoxy-1,2-butanediol and hydroxymethylvinyl ketone in 3-butene-1,2-diol associated mutagenicity

1,2:3,4-Diepoxybutane is hypothesized to be the main intermediate involved in mutagenicity following exposure to low levels of 1,3-butadiene (BD) in mice, while metabolites of 3-butene-1,2-diol (BD-diol) are thought to become involved in both rats and mice at higher exposures. BD-diol is biotransformed to hydroxymethylvinyl ketone (HMVK), a potentially mutagenic metabolite, and 3,4-epoxy-1,2-butanediol (EB-diol), a known mutagen. To determine the relative importance of HMVK and EB-diol in BD-diol associated mutagenesis, we have examined the dosimetry of a HMVK derived DNA adduct, as well as EB-diol derived DNA and hemoglobin adducts, in rodents exposed to BD-diol. We previously demonstrated similarities in the shapes of the dose-response curves for EB-diol derived DNA adducts, hemoglobin adducts, and Hprt mutant frequencies in BD-diol exposed rodents, indicating that EB-diol was involved in the mutagenic response associated with BD-diol exposure. To examine the role of HMVK in BD-diol mutagenicity, a method to quantify the alpha-regioisomer of HMVK derived 1,N(2)-propanodeoxyguanosine (alpha-HMVK-dGuo) was developed. The method involved enzymatic hydrolysis of DNA, HPLC purification, and adduct measurement by liquid chromatography - tandem mass spectrometry. Intra- and inter-experimental variabilities were determined to be 2.3-18.2 and 4.1%, respectively. The limit of detection was approximately 5 fmol of analyte standard injected onto the column or 5 fmol/200 microg DNA. The method was used to analyze liver DNA from control female F344 rats and female F344 rats exposed to 36 ppm BD-diol. In addition, liver samples from female Sprague-Dawley rats exposed to 1000 ppm BD were analyzed. alpha-HMVK-dGuo was not detected in any of the samples analyzed. Several possible explanations exist for the negative results including the possibility that alpha-HMVK-dGuo may be a minor adduct or may be efficiently repaired. Alternatively, HMVK itself may be readily detoxified by glutathione (GSH) conjugation. While experiments must be conducted to understand the exact mechanism(s), these results, in addition to published EB-diol derived adduct dosimetry and existing HMVK derived mercapturic acid data, suggest that EB-diol is primarily responsible for BD-diol induced mutagenicity in rodents.     

Cryopreservation of human platelets with propane-1,2-diol

The preceding papers in this series have described techniques that permit the introduction and removal of propane-1,2-diol (propylene glycol, PG) with human platelets, in concentrations up to 2 M, without producing serious damage. These methods have now been used in attempts to cryopreserve platelets, with assessment of survival by the hypotonic stress response and ADP-induced aggregation. PG concentrations of 0.5, 1.0, 2.0, and 2.5 M and cooling rates between 0.4 and 100 degrees C/min were studied. The maximum response in the hypotonic stress test was no better than 17% and the greatest ADP-induced aggregation was only 6%; these results were obtained with 0.5 M PG, a cooling rate of 14 degrees C/min, and rapid warming (approximately 150 degrees C/min). The failure of PG concentrations greater than 0.5 M to improve survival was unexpected. When cooling was interrupted at progressively lower temperatures and function assessed, it was possible to relate the extent of damage to temperature and then, with the aid of phase diagrams, it was possible to show that, irrespective of the initial concentration of PG, the extent of damage was closely correlated with the concentration of PG produced at the minimum temperature used. It is concluded that the toxicity of PG increases so steeply with the increasing concentration produced by the separation of ice during freezing that this effect is sufficient to counteract the cryoprotective action of this solute for platelets.     

Permeation of glycerol and propane-1,2-diol into human platelets

The permeability of human platelets to glycerol (GLY) and propane-1,2-diol (propylene glycol, PG) has been determined by measuring the time course of their change in volume following abrupt immersion in solutions of these solutes. A simple light-scattering method, and its calibration to measure mean platelet volume is described. The data are analyzed by means of the Kedem-Katchalsky (K-K) equations, modified to take into account the nonideal behavior of both intracellular and extracellular solutes. The values of the K-K parameters at 2, 21, and 37 degrees C, respectively, were as follows: the hydraulic conductivities (Lp) were 1 x 10(-7), 7 x 10(-7) and 3 x 10(-6) cm.sec-1.atm-1; the solute permeabilities for PG (omega RTPG) were 1.9 x 10(-6), 2.8 x 10(-5), and 1.3 x 10(-4) cm.sec-1; the solute permeabilities for GLY (omega RTGLY), at 21 and 37 degrees C only, were 2.6 x 10(-7) and 1.4 x 10(-6) cm.sec-1. The reflection coefficient (sigma) was 1 throughout. The relevant activation energies were -Lp, 16.5 kcal.mol-1; omega RTPG, 20.5 kcal.mol-1; and omega RTGLY, 17.9 kcal.mol-1. The use of these data is illustrated by computing schedules for the addition and removal of GLY and PG so that the amplitudes of changes in platelet volume are held within predetermined limits.     

Corneal tolerance of vitrifiable concentrations of propane-1,2-diol

The merit of corneal cryopreservation by vitrification as opposed to conventional freezing is the avoidance of ice damage which is believed to disrupt the integrity of the corneal endothelium resulting in loss of corneal transparency. The cornea must be equilibrated with high concentrations of cryoprotectant in order to achieve vitrification at practicable cooling rates. In an earlier study, corneas were exposed to 3.4 mol/liter propane-1,2-diol (Rich and Armitage (1990) Cryobiology 27, 42-54). The present study exposed rabbit corneas to concentrations of propane-1,2-diol between 3.4 and 5.4 mol/liter in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, 6% (w/v) bovine serum albumin, and 2.5% (w/v) dextran sulfate. Dextran sulfate was as effective as chondroitin sulfate at improving endothelial tolerance of 3.4 mol/liter propane-1,2-diol. This beneficial effect may be linked to the polyanionic nature of these molecules. Corneas exposed to 5.4 mol/liter propane-1,2-diol were cooled in liquid nitrogen vapor at a temperature of -140 degrees C for 2 h. Warming was achieved by direct transfer to a dilution solution at -10 degrees C. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial structure was observed by specular microscopy during the perfusion and by scanning electron microscopy after perfusion. Corneas tolerated exposure to 3.4 mol/liter propane-1,2-diol for 20 min at 0 degrees C and to 4.1 mol/liter for 10 min at -10 degrees C. Exposure to 4.8 and 5.4 mol/liter for 10 min at -10 degrees C caused endothelial damage, although a degree of endothelial function was retained. Function following exposure to 5.4 mol/liter was improved by reducing the temperature of exposure to -15 degrees C. Corneas cooled after exposure to 5.4 mol/liter propane-1,2-diol for 10 min at -15 degrees C apparently vitrified, but devitrified on warming. The corneas swelled to such an extent during perfusion that the endothelium could not be viewed by specular microscopy, subsequent scanning electron microscopy showed a severely disrupted endothelium.     

Some effects of propane-1,2-diol on human platelets

The Kedem-Katchalsky equations and permeability data previously reported (F. G. Arnaud and D. E. Pegg. Permeation of glycerol and propane-1,2-diol into human platelets. Cryobiology 27, 130-136, 1990) have been used to design methods for adding and removing propane-1,2-diol (propylene glycol, PG) with human platelets. Mean platelet volume was kept within the tolerated range of 60 to 120% of normal. PG concentrations of 0.5, 1.0, 2.0, 2.5, and 3.0 M were studied at 2, 21, and 37 degrees C. PG was removed only at 21 degrees C. The effects of concentration of PG, temperature, and duration of exposure on the hypotonic stress response and ADP-induced aggregation were measured. It was found that platelets would tolerate exposure to PG concentrations up to 2 M at 21 or 2 degrees C for up to 15 min. The extent of damage increased considerably at higher temperatures and concentrations. These data provide the necessary basis for experiments to cryopreserve platelets with PG.     

Biological Process for Converting Naphthalene to cis-1,2-Dihydroxy-1,2-Dihydronaphthalene

A biological process for converting naphthalene to cis-1,2-dihydroxy-1,2-dihydronaphthalene (DHD) catalyzed by Pseudomonas putida strain 119 was optimized in flask experiments. These studies revealed the following: (i) P. putida 119 can propagate efficiently and produce DHD when supplied one of several carbon sources and naphthalene; (ii) maximum DHD production by P. putida 119 occurs in logarithmic-growth-phase cells and decreases at various rates in the stationary growth phase, depending upon the carbon source used; (iii) several analogs of salicylic acid can be used as effective inducers of naphthalene metabolism in P. putida cells growing on glucose; and (iv) the addition of chemical surfactants to naphthalene-cell (P. putida 119) mixtures stimulates DHD production.     

A new bacterial sphingophospholipid containing 3-aminopropane-1,2-diol

1. A new sphingophospholipid has been isolated from the bacterial fraction of sheep rumen contents. 2. This new lipid has been characterized as a ceramide phosphate moiety esterified to 3-aminopropane-1,2-diol through the primary alcohol group. 3. Mass spectrometry has shown the intact lipid to contain a vicinal hydroxyl amino grouping, and oxidation with periodate converts it quantitatively into a new phospholipid which is probably ceramide phosphorylglycolaldehyde. 4. The sphingophospholipid contains a mixture of saturated long-chain bases mainly with branched-chain alkyl groups, which are typical of a microbial origin. 5. A Gram-negative bacterium isolated from rumen contents contains about 30% of the new lipid in its phospholipids when grown in pure culture.     

Oxidation of trans- and cis-1,2-cyclohexanediol by Gluconobacter oxydans

Summary The enzymatic oxidation of 1,2-cyclohexanediol and related substrates by Gluconobacter oxydans (ATCC 621) was investigated. At low pH, membrane-bound enzymes were active and at high pH, NAD-dependent, soluble enzymes showed activity. Whole bacterial cells were used to catalyze some bioconversions. Racemic trans-1,2-cyclohexanediol was oxidized at pH 3.5 to give (R)-2-hydroxycyclohexanone (96% e.e.) and at pH 8.0 the same substrate was oxidized to (S)-2-hydroxycyclohexanone (97% e.e.). The latter conversion was severely inhibited by the reaction product while the former was not significantly product inhibited. (S)-2-hydroxycyclohexanone (97% e.e.) was also prepared from cis-1,2-cyclohexanediol by oxidation with G. oxydans cells at pH 3.5 in a reaction which continued to 100% conversion.     

Perfusion of rabbit kidneys with solutions containing propane-1,2-diol

Propane-1,2-diol (propylene glycol, PG) permeates more rapidly than glycerol, has a strong glass-forming tendency, and appears to have a low toxicity. It is therefore attractive as a potential cryoprotectant for renal preservation. In this paper we compared the effect on subsequent function, of exposing rabbit renal cortical slices to 1 M PG or glycerol in a range of vehicle solutions and we demonstrated a remarkably low toxicity of PG at this concentration. Rabbit kidneys were then perfused with solutions containing PG up to a maximum concentration of 3 M, after which the cryoprotectant was removed and the function of cortical slices prepared from the perfused kidneys was assessed. Marked differences in perfusion characteristics were found between PG and glycerol and between different vehicle solutions for PG, but the two most suitable perfusates, both containing about 100 mM mannitol, permitted normal function in slices prepared after removal of PG. These results indicate that, with an appropriate vehicle perfusate, exposure to PG up to a concentration of 3 mol/liter has remarkably little effect upon vascular resistance and the renal cortical functions measured.     

Anaerobic degradation of cyclohexane-1,2-diol by a new Azoarcus species

A bacterium, strain 22Lin, was isolated on cyclohexane-1,2-diol as sole electron donor and carbon source and nitrate as electron acceptor. Cells are motile rods and are facultatively anaerobic. A phylogenetic comparison based on the total 16S rRNA gene sequence allowed the assignment of the isolate to the genus Azoarcus. Cyclohexanol, cyclohexanone, cyclohexane-1,3-diol, and cyclohexane-1,3-dione, which are oxidized by a different denitrifying strain, did not support denitrifying growth of isolate 22Lin. On the contrary, cyclohexanol (I₅₀ = 37 μM) and cyclohexanone (I₅₀ = 28 μM) inhibited growth on cyclohexane-1,2-diol, but not on acetate. NAD was reduced by crude extracts of strain 22Lin in the presence of cyclohexane-1,2-dione, but not in the presence of cyclohexanone or cyclohexane-1,3-dione. The formation of 6-oxohexanoate from cyclohexane-1,2-dione and of adipate during NAD reduction suggests that strain 22Lin possesses a carbon–carbon hydrolase that transforms cyclohexane-1,2-dione into 6-oxohexanoate. This pathway was once observed in an aerobic pseudomonad that was lost and could not be reisolated. Here, the application of strictly anoxic enrichment conditions enabled the reisolation of another strain (22Lin) that uses this pathway. Received: 3 February 1997 / Accepted: 12 May 1997     

Anthranilic acid and 3-indolyl-propan-1,2-diol as precursors of photorubin in Saccharomyces

Summary The formation of photorubin, the orange-red pigment produced by Saccharomyces, is due to a photochemical reaction between anthranilic acid and an indole derivative identified with 3-indolyl-propan-1,2-diol. These two compounds are excreted into the medium by strains partly or completely requiring pyridoxine which are grown in the presence of a suboptimal amount of this vitamin. If pyridoxine is added to the growth medium in amounts either sufficient or exceeding the needs of the cells (over 50 g per liter), or if the strain is auxo-autotrophic, a secretion of hypoxanthine occurs.