Production of glycerol by Lactobacillus plantarum NRRL B-4496 and formation of hexamine during fermentation of pea protein enriched flour.

Suspensions of pea protein enriched flour (PP) inoculated with Lactobacillus plantarum NRRL B-4496 and uninoculated PP suspensions were incubated in vials covered with airtight caps. Organic compound compositions of fermented and unfermented PP suspensions (F-PP and U-PP, respectively) were analyzed using solid phase microextraction (SPME) coupled with gas chromatography - mass-spectrometry (GCMS). Acetic acid was detected in all samples; pH dropped from pH 6.5 to pH 4.1 in L. plantarum F-PP and to pH 5.3 in uninoculated F-PP. Abundance of acetic acid and minuscule presence of lactic acid in L. plantarum F-PP suggested that fermentation proceeded preferentially via the pyruvate formate lyase (PFL) pathway. Nonetheless, glycerol appeared to be the most abundant compound in L. plantarum F-PP samples; colorimetric analysis indicated that its average concentration in these samples was 1.05 g/L. A metabolic switch from the PFL pathway to glycerol production might occur due to acidity tolerance limitations of L. plantarum, glycerol production being associated with the release of phosphate, which can act as a buffer. Fermentation of PP by L. plantarum also led to formation of hexamine, which is a known food preservation agent. Presence of naturally formed hexamine and glycerol in food products may render using chemical additives needless.

Methenamine's journey of 160 years: Repurposal of an old urinary antiseptic for treatment and hypoxic radiosensitization of cancers and glioblastoma.

Methenamine (hexamethylenetetramine, hexamine, urotropine) is a compound discovered in 1859, which is still currently being used as a urinary antiseptic. Methenamine is highly soluble in water and polar solvents, and its molecular constitution is similar to adamantane compounds with tetrahedral cage like structure. In acidic conditions, methenamine decomposes to formaldehyde and ammonia. Recently, methenamine has gained a renewal of interest due to antibiotic-resistant bacteria urinary tract infections; interestingly, bacteria cannot gain resistance to formaldehyde. In 1968, David and Burkitt reported remarkable regression of four Burkitt Lymphoma patients in eight subjects who were treated with septicemine (a solution containing 6.3 g of methenamine iodomethylate and 1 g of methenamine sodium benzoate in 100 cc distilled water). Unfortunately, these striking observations did not gain interest in the medical community; despite experimental models that showed that methenamine synergized with hyperthermia, radiation, and chemotherapy to block cancer growth. As the hypoxic core of tumours have an acidic pH, it would be plausible to expect that methenamine would selectively target dormant, non-proliferative, and treatment-resistant cancer clones in large tumours. Moreover, previous data suggests that methenamine can be safely used intravenously and for treatment of infections of the central nervous system. It may therefore be an effective adjuvant in treatment of systemic cancers and glioblastoma.

A pH-sensitive methenamine mandelate-loaded nanoparticle induces DNA damage and apoptosis of cancer cells.

Methenamine mandelate is a urinary antibacterial agent, which can be converted to formaldehyde in urine that has a relatively low pH of average 5.5-6.8. Here, we prepare a pH-sensitive PLGA-based nanoparticle containing both methenamine mandelate and NaHCO3. Methenamine mandelate/NaHCO3-coloaded nanoparticle could enter cells via endosome/lysosome pathway. The pH in lysosomes and endo-lysosomes is approximately 5.0. In the acidic environment, NaHCO3 reacts with proton and produce CO2 bubbles, which burst nanoparticles and lead to the rapidly release of methenamine mandelate. Meanwhile, methenamine mandelate was then quickly converted to a sufficient amount of formaldehyde in this acidic environment, which induced DNA damage and DNA damage response (DDR). Consequently, methenamine mandelate/NaHCO3-coloaded nanoparticles caused cell cycle arrest, cell growth inhibition and apoptosis of cancer cells. Moreover, methenamine mandelate/NaHCO3-coloaded nanoparticles also show intensive inhibitory effect on the growth of MCF-7 xenograft tumor in vivo. Therefore, methenamine mandelate/NaHCO3-coloaded nanoparticle is a promising type of formulation for the treatment of cancer, which could give the "old drug" methenamine mandelate a new anti-cancer function in clinical. STATEMENT OF SIGNIFICANCE: Methenamine mandelate is a urinary antibacterial agent, which can be converted to formaldehyde in urine that has a relatively low pH of average 5.5-6.8. Here, we prepare a pH-sensitive PLGA-based nanoparticle containing both methenamine mandelate and NaHCO3. Methenamine mandelate/NaHCO3-coloaded nanoparticle could enter cells via endosome/lysosome pathway. The pH in lysosomes and endo-lysosomes is approximately 5.0. In the acidic environment, NaHCO3 reacts with proton and produce CO2 bubbles, which burst nanoparticles and lead to the rapidly release of methenamine mandelate. Meanwhile, methenamine mandelate was then quickly converted to a sufficient amount of formaldehyde in this acidic environment, which induced DNA damage and DNA damage response (DDR). Methenamine mandelate/NaHCO3-coloaded nanoparticle is a promising type formulation for the treatment of cancer, which could give the "old drug" methenamine mandelate a new anti-cancer function in clinical.

Attempts to counteract phosgene-induced acute lung injury by instant high-dose aerosol exposure to hexamethylenetetramine, cysteine or glutathione.

Phosgene is an important high-production-volume intermediate with widespread industrial use. Consistent with other lung irritants causing ALI (acute lung injury), mode-of-action-based countermeasures remain rudimentary. This study was conducted to analyze whether extremely short high-level exposure to phosgene gas could be mitigated using three different inhaled nucleophiles administered by inhalation instantly after exposure to phosgene. Groups of young adult male Wistar rats were acutely exposed to carbonyl chloride (phosgene) using a directed-flow nose-only mode of exposure of 600 mg/m³ for 1.5 min (225 ppm × min). Immediately after exposure to phosgene gas the rats were similarly exposed to three strong nucleophiles with and without antioxidant properties for 5 or 15 min. The following nucleophiles were used: hexamethylenetetramine (HMT), l-cysteine (Cys), and l-glutathione (GSH). The concentration of the aerosol (mass median aerodynamic diameter 1.7-2 µm) was targeted to be in the range of 1 mg/L. Cys and GSH have antioxidant properties in addition. The calculated alveolar molar dosage of phosgene was 9 µmol/kg. At 15-min exposure duration, the respective inhaled dose of HMT, Csy, and GSH were 111, 103, and 46 µmol/kg, respectively. The alveolar dose of drugs was ~10-times lower. The efficacy of treatment was judged by protein concentrations in bronchoalveolar lavage fluid (BALF) collected 1 day post-exposure. In spite of using optimized aerosolization techniques, none of the nucleophiles chosen had any mitigating effect on BALF-protein extravasation. This finding appear to suggest that inhaled phosgene gas acylates instantly nucleophilic moieties at the site of initial deposition and that the resultant reaction products can not be reactivated even following instant inhalation treatment with competing nucleophilic agents. In spite of using maximal technically attainable concentrations, it appears to be experimentally challenging to deliver such nucleophiles to the lower respiratory tract at high dosages.

Usefulness of hexamethylenetetramine in combination with chemotherapy using free and pegylated liposomal doxorubicin in vivo, referring to the effect on quiescent cells.

SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. They received hexamethylenetetramine (HMTA) either once intraperitoneally or continuously subcutaneously together with chemotherapy using intraperitoneally administered free doxorubicin (DXR) or intravenously injected pegylated liposomal doxorubicin (PLD). One hour after the free DXR loading or 24 h after the PLD loading, the response of intratumor quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The response of the total (P + Q) tumor cell population was determined from the tumors not treated with BrdU. Encapsulation of DXR into pegylated liposomes significantly enhanced cytotoxicity, especially in Q cells. HMTA, especially when administered continuously, efficiently increased the sensitivity to DXR, particularly in Q cells. The increase in sensitivity on the continuous rather than single administration of HMTA was a little clearer in the total cell population than in Q cells. DXR's encapsulation into pegylated liposomes and combination with HMTA, particularly when administered continuously, apparently reduced the difference in sensitivity to free DXR between the total and Q cell populations. In terms of the tumor cell-killing effect as a whole, including Q cells, the encapsulation of DXR into pegylated liposomes and combination with HMTA, particularly through continuous administration, are very promising, taking into account that HMTA has been used clinically.

Antioxidative properties of natural polyamines and dimethylsilane analogues.

Structural analogues of natural polyamines, which contain a -Si(CH3)2 group in the central carbon chain, have previously been found to be cytotoxic to various tumor cell lines in vitro and to inhibit tumor cell growth in experimentally grafted animals. In the present study, the antioxidative properties of dimethylsilane polyamine analogues were analyzed in comparison with the natural polyamines. Reactivities of these various polyamines against superoxide anions (generated from the hypoxanthine/xanthine oxidase reaction) and peroxyl radicals (produced from the thermal decomposition of water-soluble 2,2'-azo-bis-[2-amidinopropane] hydrochloride) were investigated. The dimethysilane analogues, and more particularly the hexamine derivative, exhibited the highest scavenging efficiency towards these two reactive oxygen species (ROS). Furthermore, analysis of their ability to prevent hydroxyl radical formation and to trap this ROS showed that the efficiency of the hexamine as a metal chelator and hydroxyl radical scavenger is similar to that of spermine. The higher antioxidant efficiency of the dimethylsilane polyamine analogues with respect to spermidine, together with their ability to displace this polyamine, essential for the promotion of cell growth, from its cellular anionic binding sites that are particularly prone to oxidation, could be biologically relevant and contribute to their in vivo cytotoxic effect and anti-tumor activity. Further experiments will be necessary to demonstrate clearly the relationship between their antioxidant properties and their antiproliferative effects.

[Antimicrobial action of ammonium salts of fused heterocycles containing ortho-nitrogen]. / Kondensuotu heterociklu amonio drusku su azoto atomu ortopadetyje bakteriostatinis poveikis.

By alkylation of hexamethylenetetramine with halogenated derivatives of ketones, ethers, esters or amides of acids, alkyl- and aralkyl halides the corresponding N-monoalkylated compounds of hexamethylenetetramine were obtained. The quaternization of pyridine nitrogen in 5,6-benzoquinoline, 8-hydroxyquinoline, quinoline, 1,10-phenanthroline molecules with alkyl- or aralkylhalides was carried out. The susceptibility of Gram-positive (Streptococcus agalactiae and Staphylococcus aureus) and Gram-negative (E. coli, Salmonella cholerae suis, Salmonella enteridis Gartneri) microorganisms to synthesized quaternary ammonium salts by disc difussion method has been detected. The bacteriostatic action of 0.5-1% solutions of all compounds was assessed in comparison with benzalkonium chloride. It was shown, that the most effectiveness against all strains is possessed by quaternary hexamethylenetetramine ammonium salts, and especially salts, containing 1-propynyl- or hydroxycarbamoylmethyl radicals. The action of these two compounds against Salmonella and Streptococcus was stronger than the action of benzalkonium chloride. Susceptibility of Pseudomonas aeruginosa to these compounds were detected. It was shown, that 1% solutions of chlorides of N-(1-propynyl) hexamethylenetetramonium and N-(hydroxycarbamoylmethyl) hexamethylenetetrammonium demonstrate the same bacteriostatic action against P. aeruginosa as well as benzalkonium chloride.

[Quantitative assessment of the biological activity of chemicals by soil microbial associations]. / Kolichestvennaia otsenka biotsidnoi aktivnosti khimicheskikh soedinenii s pomoshch'iu mikrobnykh assotsiatsii pochvy.

Quantitative assessment, using three Pseudomonas sp. strains, of the activity of the microbial biocide Soncid 8101 demonstrated that the values of effective sublethal concentrations (L50) differed by 500% (because of individual variations in the sensitivity of the test strains). The spread of parameters of biocidal activity could be narrowed by using a mixture of microorganisms with high, medium, and weak resistance. A method for quantitative assessment of the activity of microbial biocides was proposed, based on the use of natural associations of soil bacteria.

Activation of adriamycin by the pH-dependent formaldehyde-releasing prodrug hexamethylenetetramine.

Previous studies have shown that Adriamycin can react with formaldehyde to yield an activated form of Adriamycin that can further react with DNA to yield Adriamycin-DNA adducts. Because hexamethylenetetramine (HMTA) is known to hydrolyze under cellular conditions and release six molecules of formaldehyde in a pH-dependent manner, we examined this clinical agent for its potential as a formaldehyde-releasing prodrug for the activation of Adriamycin. In IMR-32 neuroblastoma cells in culture, increasing levels of HMTA resulted in enhanced levels of Adriamycin-DNA adducts. These adducts were formed in a pH-dependent manner, with 4-fold more detected at pH 6.5 compared with pH 7.4, consistent with the known acid lability of HMTA. The resulting drug-DNA lesion was shown to be cytotoxic, with combined Adriamycin and prodrug treatment resulting in a 3-fold lower IC(50) value compared with that of Adriamycin alone. Given the acidic nature of solid tumors and the preferential release of formaldehyde from HMTA in acidic environments, HMTA therefore has some potential for localized activation of Adriamycin in solid tumors.

Polyamine structural effects on the induction and stabilization of liquid crystalline DNA: potential applications to DNA packaging, gene therapy and polyamine therapeutics.

DNA undergoes condensation, conformational transitions, aggregation and resolubilization in the presence of polyamines, positively charged organic molecules present in all cells. Under carefully controlled environmental conditions, DNA can also transform to a liquid crystalline state in vitro. We undertook the present work to examine the ability of spermidine, N4-methylspermidine, spermine, N1-acetylspermine and a group of tetramine, pentamine and hexamine analogs of spermine to induce and stabilize liquid crystalline DNA. Liquid crystalline textures were identified under a polarizing microscope. In the absence of polyamines, calf thymus DNA assumed a diffused, planar cholesteric phase with entrapped bubbles when incubated on a glass slide at 37 degrees C. In the presence of spermidine and spermine, the characteristic fingerprint textures of the cholesteric phase, adopting a hexagonal order, were obtained. The helical pitch was 2.5 micro m. The final structures were dendrimeric and crystalline when DNA was treated with spermine homologs and bis(ethyl) derivatives. A cholesteric structure was observed when DNA was treated with a hexamine at 37 degrees C. This structure changed to a hexagonal dendrimer with fluidity on prolonged incubation. These data show a structural specificity effect of polyamines on liquid crystalline phase transitions of DNA and suggest a possible physiological function of natural polyamines.

Comparison of methods for identification of Pneumocystis carinii in bronchoalveolar lavage fluid.

Pneumocystis carinii pneumonitis in one of the most common life-threatening opportunistic infections in patients with AIDS. The definitive diagnosis of this infection can be established only by demonstration of the organism in clinical specimens. This study was a comparison of methods that provide easy recognition of the organism which is readily available, simple and can be performed rapidly in laboratory-diagnosis. Bronchoalveolar lavage fluids obtained from 35 AIDS patients suspected of having Pneumocystis carinii pneumonitis were examined by three staining methods for the presence of Pneumocystis carinii. With Giemsa stains, P. carinii could be identified in 18 cases (51.4%). Three developmental stages: "cyst", "sporozoite" and "trophozoite" were seen. The contrast of organisms against host cells was not outstanding in these stains. Toluidine blue O stains provided easy recognition of the organisms, with marked contrast between the cysts and host cells. 21 cases (60%) were positive in these stains, but the intracystic structures and trophozoites could not be identified. It was suggested that the clinical specimen should be stained first with toluidine blue O which is more rapid and permits easy recognition of the cyst clusters. If the sporozoites and trophozoites had to be identified, Giemsa stains can be made. In addition, with the methenamine silver nitrate stains, 21 cases (60%) were positive. They revealed the morphology as seen with toluidine blue O but the cost of material may make it unavailable in many laboratories especially with the budgetary restraints of developing countries.

Dimethylsilane polyamines: cytostatic compounds with potentials as anticancer drugs. II. Uptake and potential cytotoxic mechanisms.

Dimethylsilane tetramines are structural analogues of spermine with a (CH3)2 Si-group incorporated into the central carbon chain. They have potential as anticancer drugs. Their cytotoxic effect was considered to rely mainly on their polyamine antagonist property. In order to obtain new ideas about cellular mechanisms, which are potential targets of the dimethylsilane polyamines, the effects of these compounds on some basic cell functions, such as protein and DNA synthesis, and calmodulin antagonism were studied. In addition, their mode of accumulation in cells was investigated. It became evident that the intracellular accumulation of dimethylsilane polyamines is almost exclusively achieved via the polyamine transport system. However, the exchange of a part of the intracellular natural polyamines against dimethylsilane polyamines has only a small effect on polyamine uptake. Binding to the endoplasmic reticulum and inhibition of protein synthesis are presumably important for the cytotoxic action of bis(11-amino-4,8-diazaundecyl)dimethylsilane, a hexamine, but seem of no importance for the tetramines. Calmodulin antagonism, however, is likely to contribute to their cytotoxic effect.

A comparison of the effect of three endodontic sealers on adherence of mouse peritoneal macrophages.

Leakage of sealer from root canals to periapical tissues during root canal obturation may occur. This study was designed to evaluate the possible effect of three root canal sealers (zinc oxide-eugenol, Grossman, and AH 26) on adherence of mouse macrophages. Macrophages were obtained from the peritoneal cavity of BALB/c mice and suspended in RPMI-1640 medium. Adherence capacity assays were carried out in Eppendorf tubes. Each sealer was tested four times after mixing (immediately, 12 hours, 24 hours, and 48 hours after) and for three period of incubation (10, 20, and 30 minutes) with suspended cells. Cells were counted under the light microscope, and the adherence index was determined. Zinc oxide-eugenol and Grossman sealers killed all macrophages, and the adherence index was considered less than 1 for these sealers. AH 26 reduced the adherence index in all different periods after mixing and incubation times. But in sealer that had mixed 48 hours before the experiment and with 10 minutes of incubation, the adherence index was increased slightly, but the difference was not statistically significant (P < 0.05).

Antimicrobial activity of four root canal sealers against endodontic pathogens.

The antibacterial effects of various types of widely used endodontic sealers have not been compared systematically on facultative or obligate anaerobic endodontic pathogens. The aim of this study was to evaluate the antimicrobial properties of four commonly used endodontic sealers: two epoxy-resin-based sealers (AH26, AH plus), one zinc-oxide eugenol-based sealer (N2), and one calcium hydroxide-based sealer (Sealapex). The testing microbes were four facultative anaerobic species (Streptococcus mutans, Streptococcus sanguis, Escherichia coli, and Staphylococcus aureus) and four obligate anaerobic species (Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum, and Prevotella intermedia). The freshly mixed sealers were placed into the prepared wells of agar plates inoculated with the test microorganisms. After varying periods of incubation (2 days for facultative anaerobic species and 7 days for obligate anaerobic species), the zones of growth inhibition were observed and measured. All the sealers were distinctly different from each other in their antimicrobial activity. The sealers showed different inhibitory effects depending on the types and bacterial strains. N2 containing formaldehyde and eugenol proved to be the most effective against the microorganisms. The extreme antimicrobial potency of this root canal sealer must be weighted against its pronounced tissue toxic effect.

Efficient protection of human bronchial epithelial cells against sulfur and nitrogen mustard cytotoxicity using drug combinations.

The aim of this study was to test the efficacy of several candidate molecules against sulfur mustard (SM) and nitrogen mustard (HN2) using a human bronchial-epithelial cell line (16HBE14o-). Candidate molecules were chosen on the basis of the known cytotoxicity mechanisms of mustards or their efficacy previously observed on other cellular models. It included the sulfhydryl-containing molecules N-acetyl-cysteine (NAC) and WR-1065, the nucleophile hexamethylenetetramine (HMT), the energy-level stabilizer niacinamide (NC), the antioxidant dimethylthiourea (DMTU), L-arginine analogues such as L-thiocitrulline (L-TC) and L-nitroarginine methyl ester (L-NAME), and the anti-gelatinase doxycycline (DOX). Their efficacy was determined using 2-(4-[3-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2Htetrazolium (WST-1) reduction by viable cells 24 h after initial exposure to 100 microM HN2 or SM. On individual immediate cotreatment, some molecules exhibited selective protection against only one mustard, such as DMTU and WR-1065 against HN2 and DOX against SM, whereas NAC and L-TC were effective against both SM and HN2 cytotoxicity. However, as the level of protection against SM was always weak compared to HN2, several combinations were investigated against SM to improve the protection. The effective combinations (L-TC + DOX, NAC + DOX, NAC + DMTU, NAC + HMT, NC + DOX) combined agents, reducing the bioavailability of the mustard with compounds possibly acting on the consequences of alkylation. One of these combinations, NAC + DOX, appeared to be the most interesting, as these agents are already used in human therapy. It exhibited good efficacy in delayed cotreatment (up to 90 min) against SM.

Effect of various mixing ratios on antibacterial properties and hardness of endodontic sealers.

Most chemically cured two-component dental materials, including endodontic sealers, are marketed with mixing instructions but with no strict mixing ratios. The present study evaluated the antibacterial properties and hardness of three endodontic sealers: Roth's cement (RC), CRCS, and AH26, mixed to four controlled consistencies within the range of the manufacturer recommendations. Using Enterococcus faecalis as the test microorganism, antibacterial activity was evaluated by agar diffusion and direct contact test. Surface hardness of sealers with the same consistency was evaluated on week-old specimens by the Knoop Hardness Number tester. In the agar diffusion test, light consistency of RC showed larger zones of inhibition than heavier consistency, whereas no significant differences were found with AH26 or CRCS. In the direct contact test, RC and CRCS exhibited complete inhibition of bacterial growth at all consistencies, whereas AH26 with the heavier consistencies did not inhibit bacterial growth at 24 h samples. The hardness of AH26 and CRCS was significantly lower with lighter consistencies. It is concluded that endodontic sealers possess different antibacterial and physical properties according to their mixing consistencies.

Gutta-percha: a look at the need for sterilization.

Many have argued that the prevention of contamination becomes a problem when gutta-percha cones are used to obturate the root canal space. This study evaluated the extent of contamination of commercially available gutta-percha cones taken directly from the manufacturer's box. Results show that if gutta-percha is not intentionally contaminated, there is no need for chemical decontamination before obturation.

Antimicrobial effect of six endodontic sealers: an in vitro evaluation.

The aim of this study was to determine the in vitro antimicrobial effect of six endodontic sealers after 2, 20 and 40 days. The sealers studied were Apexit, Endion, AH26, AH-Plus. Procosol and Ketac Endo. The microorganisms used were Candida albicans, Staphylococcus aureus, Streptococcus mutans and Actinomyces israelii. Petri dishes were filled with sterile agar and 0.1-ml wells were prepared and filled with the sealers. The agar plates were stored for 24 h at 37 degrees C. The samples were then removed, immersed in 4.5 ml of culture medium and divided into three groups. The samples in group 1 were stored for 2 days at 37 degrees C whereas the samples of groups 2 and 3 were stored at 4 degrees C for 20 and 40 days respectively. The samples were then removed and discarded, and 0.1 ml of the culture medium was seeded on the agar plates in order to perform colony forming unit counts. Apexit, Endion and AH-Plus produced slight inhibition on Streptococcus mutans at 20 days and on Actinomyces israelii at every time interval. No effect was found on Candida albicans and Staphylococcus aureus. Ketac Endo only produced an antimicrobial effect on Actinomyces israelii at 2 and 40 days. AH26 and Procosol showed antimicrobial effect at 40 days on Candida albicans, at 20 and 40 days on Streptococcus mutans and Staphylococcus aureus, and an effective inhibition on Actinomyces israelii at every time interval. Statistical analysis revealed both sealers and microorganisms to be significant factors affecting results in groups 2 and 3. In conclusion, the sealers evaluated in this study showed different inhibitory effects depending on time span. Overall, sealers containing cugenol and formaldehyde proved to be most effective against the microorganisms at the time intervals studied.

Protection of human upper respiratory tract cell lines against sulphur mustard toxicity by hexamethylenetetramine (HMT).

1. Sulphur mustard ('mustard gas', HD) is a highly toxic chemical warfare agent which affects the skin and respiratory tract. The primary targets of inhaled HD are the epithelia of the upper respiratory tract. Hexamethylenetetramine (HMT) has been shown to protect human lung cells against HD toxicity and has also been shown to be effective in vivo against the chemical warfare agent phosgene. The ability of HMT to protect against the toxicity of HD was investigated in the human upper respiratory tract cell lines BEAS-2B and RPMI 2650. 2. HD was highly toxic to both cell lines, with LC50 values of 15-30 microM. HMT, at a concentration of 10 mM, was shown to protect the cell lines against the toxic effects of 20 microM and 40 microM HD. Results demonstrated that it was necessary for HMT to be in situ at the time of exposure to HD for effective cytoprotection. No protection was seen when cells were treated with HMT following exposure to HD, or where HMT was removed prior to HD exposure. 3. Results suggest that HMT may be effective prophylaxis for exposure to HD by inhalation.