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Biomedical subjects

A H Hammond

Publications and source records attributed to A H Hammond.

16 recordsLinked to original sources
PubMed2002

Modulation of hepatocyte thiol content by medium composition: implications for toxicity studies.

Toxicity of compounds requiring glutathione for detoxification, thiol content and synthesis were determined in 24-h rat hepatocytes cultured in medium containing different concentrations of the sulphur amino acids. Glutathione synthesis was determined following prior depletion of glutathione with diethylmaleate. L-15 medium, which has high levels of cysteine and methionine (1 mM of each), provided some protection against dichloroacetone, dibromopropanol and dichloropropanol toxicity, and had a small effect on increasing glutathione content and synthesis, relative to Williams' medium E (WE) which has low levels (less than 0.5 mM) of both amino acids. However, WE containing N-acetylcysteine (NAC) (1 mM final cysteine concentration), with or without methionine (final concentration 1 mM), was a better cytoprotectant medium than L-15, markedly reducing toxicity of all three compounds, and rapidly (within 1.5 h) increasing cellular glutathione content. WE supplemented with methionine alone stimulated glutathione synthesis after an initial lag phase, and protected cultures against dichloropropanol, but not dibromopropanol or dichloroacetone, both of which are highly reactive in these cultures. There was a clear association between glutathione content at early time points in culture and toxicity observed at later time points, and overall these results indicate that differences in culture medium composition can alter intracellular glutathione content and xenobiotic toxicity.

Acetone↗
PubMed2001

Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability.

A major limitation in the construction of a functional engineered liver is the short-term survival and rapid de-differentiation of hepatocytes in culture. Heterotypic cell-cell interactions may have a role to play in modulating long-term hepatocyte behavior in engineered tissues. We describe the potential of 3T3 fibroblast cells in a co-culture system to modulate function and viability of primary isolated rat hepatocytes. Over an 18-day period after isolation, hepatocytes in pure culture rapidly declined in viability, displayed sparse bile canaliculi, and lost two function markers, the secretion of albumin and ethoxyresorufin O-dealkylase (EROD) activity. In comparison, the hepatocytes within the co-cultures maintained viability, possessed well-formed canalicular systems, and displayed both functional markers. Fixed 3T3 cells or 3T3 cell conditioned medium did not substitute for the viable 3T3 cell co-culture system in preserving hepatocyte viability and functionality.

3T3 Cells↗
PubMed1999

Effect of cyanamide on toxicity and glutathione depletion in rat hepatocyte cultures: differences between two dichloropropanol isomers.

The effect of aldehyde dehydrogenase inhibition by cyanamide pre-treatment in vitro on dichloropropanol-dependent toxicity and glutathione depletion was investigated in 24 h rat hepatocyte cultures. Cyanamide pre-treatment had no effect on nitrophenol hydroxylase, 7-methoxy-, 7-ethoxy- or 7-benzyloxyresorufin O-dealkylase activities in 24 h cultures from untreated rats, and had no effect on intracellular glutathione content in cultures from untreated rats, or in cultures from isoniazid-treated rats in which cytochrome P4502E1 (CYP2E1) is increased. In cultures from untreated animals the primary alcohol, 2,3-dichloropropanol, was not toxic and did not significantly deplete glutathione. Cyanamide pre-treatment however, potentiated both toxicity and glutathione depletion. Induction of CYP2E1 also potentiated the toxicity of 2,3-dichloropropanol, and in these cultures cyanamide pre-treatment significantly increased both toxicity and glutathione depletion. Cyanamide did not alter the toxicity or glutathione depletion due to the secondary alcohol, 1,3-dichloropropanol, irrespective of CYP2E1 induction. These results indicate that the primary alcohol isomer is metabolised to an aldehyde intermediate which depletes glutathione. Under basal conditions this metabolite appears to be effectively detoxified, but increased CYP2E1 activity and/or decreased aldehyde dehydrogenase activity promotes accumulation of metabolite, and therefore increases glutathione depletion and toxicity.

Aldehyde Dehydrogenase↗
PubMed1999

The nature of halogen substitution determines the mode of cytotoxicity of halopropanols.

The cytochrome P450-dependent generation of reactive metabolites from 1,3-dichloropropanol and 1,3-dibromopropanol was assessed in a microsomal thiol depletion assay, while the toxicity of these compounds was assessed in rat hepatocyte cultures and in the 3T3 cell line. Thiol-depleting metabolites of both compounds were generated in the microsomal assay; however, only dibromopropanol extensively depleted glutathione when glutathione S-transferase was used as the enzyme source. The cytotoxicity of dichloropropanol was both cytochrome P450- and glutathione-dependent, whereas that of dibromopropanol was glutathione-dependent but largely independent of cytochrome P450. These results indicate that the mechanisms underlying the cytotoxicity of halopropanols are dependent on the nature of the halogen substitution and that microsomal and cellular assays for reactive metabolite generation may yield conflicting results.

3T3 Cells↗
PubMed1997

Involvement of cytochrome P4502E1 in the toxicity of dichloropropanol to rat hepatocyte cultures.

Hepatocytes were isolated and cultured from untreated rats and rats treated with isoniazid to induce cytochrome P4502E1. Isoniazid selectively increased p-nitrophenol hydroxylase activity in 2-h cultures, and increased the toxicity of both 1,3- and 2,3-dichloropropanol. Isoniazid also increased the rate and extent of glutathione depletion by the dichloropropanols. There was no effect of isoniazid on the toxicity of 1,3-dichloroacetone, precocene II or allyl alcohol. In addition, diethyldithiocarbamate selectively inhibited p-nitrophenol hydroxylase in 2-h cultures from untreated and isoniazid-treated rats, as well as abolishing toxicity of the dichloropropanols. In 24-h cultures from isoniazid-treated rats diethyldithiocarbamate inhibited high affinity MCOD activity by 55% and there was also a small but significant inhibition of precocene II toxicity. These results indicate that isoniazid-inducible P4502E1 can mediate the toxicity of dichloropropanol.

1-Propanol↗
PubMed1995

Mechanism of toxicity of precocene II in rat hepatocyte cultures.

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 microM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 microM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na+/K+-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway.

Adenosine Triphosphate↗
PubMed1994

Comparison of in vivo and in vitro rat hepatic toxicity of coumarin and methyl analogues, and application of quantitative morphometry to toxicity in vivo.

The rat hepatic toxicity of coumarin and methyl analogues (3-,4-methyl coumarin and 3,4-dimethylcoumarin) has been determined in vivo and in vitro (freshly-isolated cells). Coumarin at a dose of approximately 1 mmol/kg produced clear histological evidence of centrilobular necrosis, while the methyl analogues at an equivalent dose were much less toxic. By use of a systematic random sampling protocol and quantitative morphometry it was determined that there was a lobar variation in the extent of hepatic damage but that this exhibited random inter-animal variation. The order of cytotoxicity in vitro was identical to that observed in vivo. In hepatocytes depleted of glutathione the toxicity of all four compounds was increased. This was particularly marked for the 3-methyl analogues, such that the order of toxicity was different to that observed in vivo and in hepatocytes not depleted of glutathione.

Animals↗
PubMed1994

Toxicity of precocene II in rat hepatocyte cultures: effects of serum and culture time.

Omission of serum from culture medium markedly increased the toxicity of the cytochrome P450-activated toxin precocene II in 24-h cultures of rat hepatocytes. In addition, the depletion of glutathione and inhibition of cytochrome P450 in 24-h cultures increased and decreased, respectively, the toxicity of precocene II, as seen in vivo. Precocene II was also toxic to 72-h cultures, although the effect of the serum-free medium was attenuated. However, depletion of glutathione and inhibition of P450 had little or no effect on the toxicity in 72-h cultures. Therefore, the mechanism of precocene II-induced cell death appears to change with time in culture.

Animals↗
PubMed1993

Maintenance of xenobiotic metabolism and toxicity in rat hepatocyte cultures after cell preservation at 4 degrees.

Isolated rat hepatocytes were stored at 4 degrees in modified University of Wisconsin solution for 22-24 hr and then placed into culture. Satisfactory viabilities were obtained for all preparations. No loss of alcohol dehydrogenase or 7-ethoxy-3,4-dimethylcoumarin O-deethylase activity was observed in the cold stored relative to the control cultures. The protein and glutathione contents, allyl alcohol and precocene II (PII) toxicities were also similar, as was the PII-mediated glutathione depletion. For the parameters measured, cultures of cold stored hepatocytes were identical to normal cultures.

1-Propanol↗
PubMed1992

Effect of serum-free medium on cytochrome P450-dependent metabolism and toxicity in rat cultured hepatocytes.

Cytochrome P450 (P450)-dependent activities in homogenates of rat hepatocytes cultured for 96 hr in serum-free and serum-containing medium were compared. Benzphetamine and erythromycin N-demethylases, 7-methoxy- and propoxy-coumarin O-dealkylases, p-nitrophenol hydroxylase and 7-ethoxy- and pentoxy-resorufin O-dealkylases were all maintained at higher levels in hepatocytes cultured in serum-free medium, although there was some selectivity with respect to the extent of the maintenance relative to the activities in fresh cells. The toxicities of coumarin, precocene I and precocene II to 24 hr hepatocyte cultures, determined as decreased survival, were also shown to be increased in serum-free medium. However, the magnitude of the difference between media with respect to the toxicity of precocene II was decreased in hepatocytes cultured for 72 hr. The observed increase in toxicity is consistent with the improved maintenance of P450 in hepatocytes cultured in serum-free medium, although there is still a selective decline in P450 activities and toxicity with increased time in culture. The activity of alcohol dehydrogenase and the toxicity of allyl alcohol were similar in hepatocytes cultured in serum-free and serum-containing medium for 96 hr. The absence of serum did not affect the non-protein sulphydryl content of the cultures.

1-Propanol↗
PubMed1991

Lupus erythematosus profundus with partial C4 deficiency responding to thalidomide.

A female patient with disfiguring lupus erythematosus profundus (LEP) from the age of 13 years was found to have an isolated partial C4 deficiency, with reduced levels of both allotypes, C4A and C4B. A genetic basis for the hypocomplementaemia was confirmed by a family study of complement and HLA types which revealed heterozygous null alleles for C4A and C4B in the proband. Marked improvement in her cutaneous lesions occurred with thalidomide.

Adolescent↗
PubMed1991

Maintenance of monooxygenase activities and detection of cytochrome P-450-mediated cytotoxicity in Mongolian gerbil hepatocyte cultures.

1. Hepatocytes were isolated from untreated and phenobarbitone (PB)-treated Mongolian gerbils by lobe perfusion. Yields were approx. 20 x 10(6) cells/g liver and viability was 95 +/- 1%. 2. PB treatment significantly increased the total cytochrome P-450 content, and the 7-ethoxycoumarin O-deethylase, p-nitrophenol hydroxylase and coumarin 7-hydroxylase activities, relative to those of untreated gerbils, measured in homogenates of freshly isolated hepatocytes. 3. After 24 h in culture the cytochrome P-450 content of hepatocyte homogenates from both untreated and PB-treated gerbils was 40-45% that of the corresponding values of freshly isolated hepatocytes. This decrease was accompanied by selective losses of cytochrome P-450-dependent enzyme activities. 4. Erythromycin and benzphetamine N-demethylase, and p-nitrophenol hydroxylase, activities were well maintained over 24 h in culture, whilst 7-ethoxycoumarin O-deethylase and coumarin 7-hydroxylase activities were poorly maintained. In general, the stability of the monooxygenase activities measured was improved by BP treatment of gerbils. 5. The toxicity of coumarin, precocene I and precocene II to gerbil hepatocyte cultures was dose-dependent. Precocene II was significantly more toxic to hepatocytes cultured from PB-treated, compared with untreated, gerbils. 6. Gerbil hepatocyte cultures would seem to be appropriate for investigating species differences in metabolism-mediated cytotoxicity.

Animals↗
PubMed1990

The in vivo induction of rat hepatic cytochrome P450-dependent enzyme activities and their maintenance in culture.

Cytochrome P450-dependent enzyme activities were measured in hepatocytes from adult male rats, induced in vivo with phenobarbitone, beta-naphthoflavone, dexamethasone or isoniazid: the stability of the induced activities in culture was also determined. Each inducer produced a characteristically different pattern of enzyme activities with dexamethasone, isoniazid and beta-naphthoflavone selectively inducing erythromycin N-demethylase, p-nitrophenol hydroxylase and ethoxyresorufin O-dealkylase respectively. In general, the induced activities were maintained for 24 hr in culture. This indicates the feasibility of an in vivo induction-hepatocyte culture system for the study of metabolism-mediated toxicity.

Animals↗
PubMed1978

Understanding rheumatoid arthritis. Evaluation of a patient education program.

We evaluated a patient education program on understanding rheumatoid arthritis; 94% of those responding to our questionnaire considered the program helpful in increasing their understanding of their condition, and 93% said it enhanced their communication with their family. A total of 85% reported one or more behavioral changes, such as not abusing joints (63%), getting more rest (56%), and using medications more meticulously (48%) after participating in the program. We recommend patient education as an effective means of helping patients understand and hence comply with physicians' instructions as well as helping patients to assume greater responsibility for their own health care.

Arthritis, Rheumatoid↗