Primary cultures and the levels of cytochromeP450 in hepatocytes from mouse, rat, hamster, and rabbit liver

Maslansky, Carol J.; Williams, Gary M.

doi:10.1007/bf02796423

Cited by 129 publications

(44 citation statements)

References 56 publications

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“…9-11) These activi- ties are usually markedly decreased after the start of a primary culture of hepatocytes. 30,31) The present investigation quantitatively confirmed a rapid decline in the mRNA expression of these P450 s. In contrast, the expression of the mRNA of UGT1A6 and SULT1A1 recovered to approximately 40% of that in vivo with an increasingly longer culture period. This evidence indicates a deficiency on the primary culture of hepatocytes, which does not precisely reflect the situation in vivo.…”

Section: Discussionsupporting

confidence: 77%

Alteration of Acetaminophen-induced Cytotoxicity in Mouse Hepatocytes during Primary Culture

Jaruchotikamol

Takase

Itoh

et al. 2009

JOURNAL OF HEALTH SCIENCE

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The present study investigated issues regarding cytotoxicity tests using mouse hepatocytes in primary culture. The results indicated that the cytotoxicity of acetaminophen (APAP) markedly varied depending on culture period for up to 5 days. P450 isoforms involved in the activation of APAP, such as CYP1A2, CYP2E1, and CYP3A, showed a marked decrease in expressions of respective mRNA. Their expression levels were a few percent within 24 hr after the start of the culture, and remained low throughout. Meanwhile, the mRNA expression of enzymes involved in the detoxification of APAP, UGT1A6 and SULT1A1, declined moderately and then recovered to approximately 40% of the in vivo level later in the culture. Treatment with APAP at non-cytotoxic concentrations increased expressions of CYP1A2, CYP2E1, CYP3A11 and CYP3A41 mRNA, while it decreased that of SULT1A1 mRNA. Using the lactate dehydrogenase (LDH) assay, the cells treated with 25-mM APAP for 24-48 or 48-72 hr showed higher cytotoxicity values than those for 72-96 or 96-120 hr, but the values after 50-mM treatment were completely inverted. By employing the methylthiazolyl tetrazolium (MTT) assay, culture period-dependent cytotoxicity was also observed on 25-mM APAP treatment, but this was not clear at 50 mM. Lower LDH or higher MTT activities than the control were observed in cells treated with lower concentrations of APAP, becoming the more prominent the longer the culture period. The observations suggested that, since the expression of metabolizing enzymes alters markedly during the primary culture and the range of cytotoxic concentrations of a test compound varies depending on the culture period, it is necessary to take account of these fluctuating parameters when assessing the cytotoxic character of a compound.

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Section: Discussionsupporting

confidence: 77%

Alteration of Acetaminophen-induced Cytotoxicity in Mouse Hepatocytes during Primary Culture

Jaruchotikamol

Takase

Itoh

et al. 2009

JOURNAL OF HEALTH SCIENCE

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“…The extent of the EndoG expression was decreased as the incubation time was increased after the transfection of EndoG siRNA (date not shown). Incubation time is known to be an important determinant affecting the outcomes of experiments, especially those using the primary culture of hepatocytes (68,69). In fact, we found that incubation times longer than 14 h before ATZϩMS treatment caused significant differences in some parameters compared with those at 2 h of incubation (date not shown).…”

Section: Discussionmentioning

confidence: 74%

Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress

Ishihara

Shimamoto

2006

Journal of Biological Chemistry

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We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ؉MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease (DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mM. ATZ؉MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ؉MS.

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“…Liver cell culture is a popular model system to study mechanisms of hepatotoxicity in experimental animals (for reviews see 1 and 2). However, a limitation of this model is the rapid loss of cytochrome P-450 during the first 24 hours of hepatocyte culture (3)(4)(5). This is an obstacle since cytochrome P-450 mediated metabolism frequently determines the toxicity and carc~nogenicity of many natural as well as synthetic chemicals (6).…”

Section: Introductionmentioning

confidence: 99%

The concentration of cytochrome P-450 in human hepatocyte culture

Blaauboer

Holsteijn

Graft

et al. 1985

Biochemical Pharmacology

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INTRODUCTIONA problem with the study of drug metabolism and toxicity in experimental animals is extrapolating the relevance of the results to man. It is unethical to determine the toxic properties of chemicals by dosing these to humans but cell cultures prepared from human tissues could circumvent this problem. Liver cell culture is a popular model system to study mechanisms of hepatotoxicity in experimental animals (for reviews see 1 and 2). However, a limitation of this model is the rapid loss of cytochrome P-450 during the first 24 hours of hepatocyte culture (3)(4)(5). This is an obstacle since cytochrome P-450 mediated metabolism frequently determines the toxicity and carc~nogenicity of many natural as well as synthetic chemicals (6).In rat hepatocyte culture the loss of cytochrome P-450 can be prevented by including metyrapone or 1,2-cyanophenylimidazole (7) in the culture medium. Little is known about the effect of the culture conditions on the concentration of cytochrome P-450 in human hepatocytes and where results have been reported these are only on a few donors (8-10). The present study reports our experience with hepatocyte cultures prepared from seven human individuals and therefore, to the best of our knowledge, represents the largest study to be reported, The results show that, in contrast to hepatocyte cultures prepared from experimental animals (3,4) the majority (i.e. five) of the cultures prepared from human liver do not lose cytochrome P-450 during the first 24 hours of culture. Furthermore, where the loss of cytochrome P-450 did occur in culture the addition of metyrapone or 1,2-cyanophenylimidazole to the culture medium, which prevents the loss of the cytochrome in rat hepatocyte culture (7), also prevents the loss of the cytochrome in cultured human hepatocytes. MATERIALS AND METHODSIsolation of hepatocytes. Liver samples were obtained from renal transplant donors who had met traumatic death and were maintained on life support systems until the kidneys were removed. Donors were not receiving any chronic drug treatment but were given such drugs as necessary while on life support systems. After removal of the kidneys a section of liver (approximately 100 g) was removed from the organ such that the section had only one cut face. In order to remove as much of the entrapped blood as possible, the liver section was perfused with Hank's balanced salt solution (HBSS from Gibco Europe) via the larger vessels on the cut surface, using a 20 ml syringe and a flow rate of ca 20 ml/min. The procedure to isolate hepatocytes commenced within 30 minutes of removal of the liver section from the donor. A smaller section (approx 30 g) was cut, ensuring that the section had only one cut face and this piece was used for the isolation of hepatocytes. A vessel was selected, so that a cannula with a male luer end-piece fitted snugly into it. Too large a vessel resulted in leakage of perfusate, too small a vessel resulted in the vessel being damaged and both resulted in poor perfusion. The liver section was perfuse...

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Primary cultures and the levels of cytochromeP450 in hepatocytes from mouse, rat, hamster, and rabbit liver

Cited by 129 publications

References 56 publications

Alteration of Acetaminophen-induced Cytotoxicity in Mouse Hepatocytes during Primary Culture

Alteration of Acetaminophen-induced Cytotoxicity in Mouse Hepatocytes during Primary Culture

Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress

The concentration of cytochrome P-450 in human hepatocyte culture

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