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

Hammond, Alison H.; Fry, Jeffrey R.

doi:10.1016/0006-2952(90)90567-5

Cited by 22 publications

(4 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The low level of DPD activity observed in human colon carcinoma cell lines suggests that down-regulation of DPD occurs in culture. This observation is similar to that seen for the cytochrome P450 enzymes, for which a lower level of enzyme activity is observed after 24-48 h in vitro (Hammond and Fry, 1990). Although in vitro studies have demonstrated a contribution of DPD in regulating the cytotoxic effect of 5-FU, the disparity between tumour and cell line data suggests that cell lines are of limited value for prediction of in vivo 5-FU activity.…”

Section: Discussionsupporting

confidence: 83%

Characterization of dihydropyrimidine dehydrogenase in human colorectal tumours

McLeod

Sludden²,

Murray³

et al. 1998

Br J Cancer

View full text Add to dashboard Cite

Summary Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme for degradation of 5-fluorouracil (5-FU). DPD activity is highly variable in liver and peripheral mononuclear cells (PMNCs) and it has not been well studied in human tumours. Characterization of DPD in colorectal cancer is of clinical interest through its role in the regulation of 5-FU, the main chemotherapeutic agent used in this disease. Therefore, DPD activity was analysed in colorectal tumour and adjacent normal tissue from 63 patients, including three liver metastasis. DPD activity was highly variable in all tissues studied (coefficient of variation 43-61 %) and was higher in normal tissue than in tumour. The tumournormal activity ratio ranged from 0.19 to 3.32 (median 0.76). PMNC DPD activity was available for 57 patients and was correlated with tumour activity (r5 = 0.29, P < 0.001). A higher correlation was observed between PMNCs and tumour samples that were both obtained in the morning (r, = 0.49), consistent with circadian variation in DPD activity. Normal tissue DPD activity was not correlated with either tumour (r5 = 0.11) or PMNC activity (rs = -0.06). This study provides the first analysis of DPD activity in colorectal cancer and illustrates the large degree of variation in tumour activity. The tumour-normal activity ratio results suggest that elevated tumour DPD can play a role in 5-FU resistance through increased inactivation in tumour cells, but is an uncommon event in colorectal tumours. The results support the use of PMNCs for monitoring tumour DPD activity, particularly when circadian variation is taken into account. As a large degree of the variation in tumour DPD activity is not explained by PMNC activity, more accurate alternatives are needed before DPD activity can be used for targeting 5-FU therapy.Keywords: dihydropyrimidine dehydrogenase; enzyme activity; colorectal cancer; 5-fluorouracil; pharmacogenetics 5-Fluorouracil (5-FU) is commonly used in the treatment of gastrointestinal, head and neck, and breast tumours. 5-FU is itself inactive and requires intracellular conversion to form cytotoxic nucleotides. Several cellular targets for fluoropyrimidines have been well characterized, including inhibition of thymidylate synthase (TS) by FdUMP and false-base incorporation into RNA or DNA. Most investigations into cellular resistance factors regulating 5-FU activity have focused on alterations in TS levels and reduced folate pools, the required cofactor for binding dUMP to TS (Wang et al, 1993;Johnston et al, 1995). However, most of an administered 5-FU dose undergoes metabolism to an inactive species through a threeenzyme process, which is initiated and rate limited by dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2). After a bolus injection of 5-FU, 80% is degraded via DPD 24 h after administration (Heggie et al, 1987). Studies of 19F-NMR spectroscopy in mice bearing colon tumours found that catabolites made up 51% of labelled drug in the tumour, compared with 26% for the anabolic products (Kamm et al, 1994). Ther...

show abstract

Section: Discussionsupporting

confidence: 83%

Characterization of dihydropyrimidine dehydrogenase in human colorectal tumours

McLeod

Sludden²,

Murray³

et al. 1998

Br J Cancer

View full text Add to dashboard Cite

show abstract

“…To measure the induction of cytochrome P4504A (CYP4A) in LiverBeads, the serumcontaining medium was replaced with serum-free medium with or without methylclofenapate, a peroxisome proliferator and inducer of CYP4A in the rat in vitro (10). Concentrations of 100µM and 312.5µM were used (allowing for the LiverBead volume, 312.5µM is equivalent to 100µM in conventional cultures), and the cells were dissociated from the alginate after 48 hours, when homogenates of the cells were prepared (11). Homogenates were subjected to SDS-PAGE (10µg protein) followed by Western blotting with a specific mouse antibody raised against a rat CYP4A1 fragment derived from a partial cDNA (12).…”

Section: Induction Of Cytochrome P4504amentioning

confidence: 99%

A Preliminary Comparison of LiverBeads™ with a Conventional Rat Hepatocyte Culture Preparation: Some Aspects of Xenobiotic Metabolism and Related Toxicity

1999

Self Cite

View full text Add to dashboard Cite

LiverBeads™ are made of hepatocytes that are immobilised and cryopreserved in alginate gel. They have great potential as an easily transportable and easily handled source of hepatocytes for use in in vitro pharmacotoxicology. In this study, we compared the drug metabolising capacity of LiverBeads in culture with that of conventional cultures and of cultures derived from cryopreserved cells. Trypan blue exclusion, lactate dehydrogenase and DNA content were measured in LiverBead cultures. The levels were all similar to those of the conventional cultures, as were the toxicities of precocene II and allyl alcohol, although more variability was seen in the LiverBeads than in the conventional cultures. The cytochrome P450-dependent activity 3,4-dimethyl-7-ethoxycoumarin-O-dealkylase, was reduced in the LiverBeads when compared to the conventional cultures, although the pattern of conjugation was very similar. In addition, the inducibility of cytochrome P4504A was demonstrated in LiverBeads. Cultures from cryopreserved cells were more susceptible to the toxicants tested, and contained less lactate dehydrogenase and DNA than the conventional cultures. Overall, in terms of the aspects of drug metabolism measured, the cultures from LiverBeads appeared to be equivalent to conventional cultures, and superior to cultures from cryopreserved cells.

show abstract

“…To reduce this complexity and as an alternative to animal and human experimentation, somatic cells were isolated and propagated in culture for biotransformation and genotoxicity studies. However, within days of establishing cultures, cells such as primary rat hepatocytes lose tissue-specific functions, especially the expression of cytochromes P450, which are the key enzymes in the metabolism of many xenobiotics [ 1,2]. In the past, metabolic deficiencies in cultivated indicator cells were overcome by using freshly prepared hepatocytes or liver homogenates as the metabolic component.…”

Section: Introductionmentioning

confidence: 99%

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17β‐estradiol and 2‐aminofluorene in V79 Chinese hamster cells

Wölfel

Platt

Dogra

et al. 1991

Molecular Carcinogenesis

View full text Add to dashboard Cite

In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.

show abstract

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

Cited by 22 publications

References 21 publications

Characterization of dihydropyrimidine dehydrogenase in human colorectal tumours

Characterization of dihydropyrimidine dehydrogenase in human colorectal tumours

A Preliminary Comparison of LiverBeads™ with a Conventional Rat Hepatocyte Culture Preparation: Some Aspects of Xenobiotic Metabolism and Related Toxicity

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17β‐estradiol and 2‐aminofluorene in V79 Chinese hamster cells

Contact Info

Product

Resources

About