Effects of Freezing, Thawing, and Storing Human Liver Microsomes on Cytochrome P450 Activity

Pearce, Robin E.; McIntyre, C J; Madan, Ajay; Sanzgiri, U. Y.; Draper, Alison J.; Bullock, Peter L.; Cook, Dan; Burton, Leroy A.; Latham, J; Nevins, Cheryl; Parkinson, Andrew

doi:10.1006/abbi.1996.0294

Cited by 216 publications

(142 citation statements)

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“…It is a good practice to check microsomal characterization data of each batch provided by the vendor with respect to cytochrome b5 and cytochrome P450 content, and NADPH-cytochrome c reductase activity, so that comparisons and justification of different study results can be made among batches. A quick and simple method to check the quality of different batches of either new or re-used microsomes is to run a UV spectrum for carbon monoxide binding difference [19,20], and if a peak at 420 nm appears, instead at 450 nm, it is likely that the CYP has started to degrade to P420, or the microsomes have been contaminated with hemoglobin that is sometimes found in the microsomes and produces a peak at 420 nm [21].…”

Section: Metabolism By Microsomesmentioning

confidence: 99%

“…Microsomes that are thawed and maintained on ice for less than 2 h can be re-frozen at −80°C and re-used without significant loss of enzyme activity [20]. This thawing-restoring flexibility allows us to use microsomal aliquots from a single batch for different experiments to facilitate normalization or comparison of different experiment data, and thus avoid confusing results introduced from different batches and vendors.…”

Section: Metabolism By Microsomesmentioning

confidence: 99%

“…A longer incubation time is not necessary [8,24]. Some vendors do not recommend for an incubation time longer than 2 h when conducting drug stability study in microsomes at 37°C [20]. If extended incubations have to be carried out for reasons, additional controls should be performed in parallel to verify that the enzyme activity is kept and no thermal degradation occurs to the test drug during the incubation period.…”

Section: Metabolism By Microsomesmentioning

confidence: 99%

See 2 more Smart Citations

The Conduct of Drug Metabolism Studies Considered Good Practice (II): In Vitro Experiments

Jia¹,

Liu²

2007

CDM

249

195

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In vitro drug metabolism studies, which are inexpensive and readily carried out, serve as an adequate screening mechanism to characterize drug metabolites, elucidate their pathways, and make suggestions for further in vivo testing. This publication is a sequel to part I in a series and aims at providing a general framework to guide designs and protocols of the in vitro drug metabolism studies considered good practice in an efficient manner such that it would help researchers avoid common pitfalls and misleading results. The in vitro models include hepatic and non-hepatic microsomes, cDNA-expressed recombinant human CYPs expressed in insect cells or human B lymphoblastoid, chemical P450 inhibitors, S9 fraction, hepatocytes and liver slices. Important conditions for conducting the in vitro drug metabolism studies using these models are stated, including relevant concentrations of enzymes, co-factors, inhibitors and test drugs; time of incubation and sampling in order to establish kinetics of reactions; appropriate control settings, buffer selection and method validation. Separate in vitro data should be logically integrated to explain results from animal and human studies and to provide insights into the nature and consequences of in vivo drug metabolism. This article offers technical information and data and addresses scientific rationales and practical skills related to in vitro evaluation of drug metabolism to meet regulatory requirements for drug development.

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Section: Metabolism By Microsomesmentioning

confidence: 99%

Section: Metabolism By Microsomesmentioning

confidence: 99%

Section: Metabolism By Microsomesmentioning

confidence: 99%

See 1 more Smart Citation

The Conduct of Drug Metabolism Studies Considered Good Practice (II): In Vitro Experiments

Jia¹,

Liu²

2007

CDM

249

195

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“…Preparation of hepatic microsomes: Hepatic microsomes were prepared as described previously [36,54]. Approximately 1-2 g of liver tissue was homogenized (Polytron Homogenizer; GTR-1000, Tokyo Rikakiki Co., Ltd., Tokyo, Japan) in a 3-volume homogenization buffer (0.1-M Tris acetate, 0.1-M KCl, 1-mM EDTA, 5 g/ml butylated hydroxytoluene, and 40 g/ml phenylmethylsulfonyl fluoride).…”

Section: Chemicalsmentioning

confidence: 99%

Detection of Initiation Activity of 1,2-Dimethylhydrazine in in vivo Medium-Term Liver Initiation Assay Ssytem using 4-Week-Old Rats without Hepatocellular Proliferative Stimuli during the Test Chemical Treatment Period

Asaoka

Sakai

Hirata

et al. 2010

The Journal of Veterinary Medical Science

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ABSTRACT. We have developed an in vivo medium-term liver initiation assay system to detect initiation activities of chemicals on multiorgan carcinogenesis. However, cell proliferation stimuli during the test chemical treatment period, required in the previously used assay models using adult rats, are laborious; moreover, those cause decrease of hepatic metabolic enzymes and psychological and physical discomfort to animals resulting in inaccurate interpretation. Therefore, we investigated the utility of another in vivo medium-term liver initiation assay model using 4-week-old rats without the cell proliferation stimuli. In this study, we confirmed that 4-week-old and 4.5-week-old male rats have high hepatocyte proliferation activity and similar enzyme activities of hepatic Cytochrome P450 subtypes as compared with 8-week-old male rats. Next, the in vivo medium-term liver initiation assay model using 4-week-old rats without cell proliferation stimuli was evaluated for the detection of the initiation activity of 1,2-dimethylhydrazine (DMH), which is a well-known genotoxic carcinogen. Four-week-old rats were orally administered DMH (single dose, 4 or 16 mg/kg; or 4-day repeat, 1 or 4 mg/kg); subsequently, these rats were treated promotion treatment consisted of administration of 2-acetylaminofluorene and carbon tetrachloride. Four weeks after the first DMH administration, the glutathione S-transferase placental form (GST-P)-positive foci induced by DMH in the liver was measured immunohistochemically. The inductions of GST-P-positive foci in all DMH-treated groups were dose-dependent, duration-dependent and significantly higher than that in non-DMH-treated group. From these results, our assay model was detected the initiation activity of DMH simply, and would be useful to evaluate the carcinogenicity of chemicals.KEY WORDS: 1,2-dimethylhydrazine (DMH)Introduction, carcinogenesis, GST-P-positive cell foci, liver, rat.J. Vet. Med. Sci. 72(1): 43-53, 2010 Several models are available today for studying the mechanism and activity of carcinogenesis in vivo, most comprising multistage studies involving initiation, promotion, and progression [15]. In vivo medium-term carcinogenic assays based on the multi-stage carcinogenesis hypothesis have been developed in order to bridge the gap between long-term in vivo carcinogenicity studies and shortterm in vitro screening tests [16,22,47,53]. The Ito model for the detection of the promotion activity in the liver has already been validated using a long list of chemicals and is widely used to detect the promotion activities of chemicals [23,24]. Initiation is also a key event in multi-stage carcinogenesis, resulting from complicated processes in vivo [17]. Therefore, an in vivo medium-term liver initiation assay model has also been developed to detect initiation activity of chemicals, and 26 well-known chemicals have been evaluated [38].The medium-term liver initiation assay model could detect genotoxic carcinogens, regardless of the target organ, with reference to the induction o...

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“…Microsomes are easy to prepare and have an excellent long term stability [6,7]. Therefore, they are a good choice to perform abundance measurements.…”

Section: Introductionmentioning

confidence: 99%

Quantification of cytochrome 2E1 in human liver microsomes using a validated indirect ELISA

Bock¹,

Colin²,

Boussery³

et al. 2014

Journal of Pharmaceutical and Biomedical Analysis

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Effects of Freezing, Thawing, and Storing Human Liver Microsomes on Cytochrome P450 Activity

Cited by 216 publications

References 0 publications

The Conduct of Drug Metabolism Studies Considered Good Practice (II): In Vitro Experiments

The Conduct of Drug Metabolism Studies Considered Good Practice (II): In Vitro Experiments

Detection of Initiation Activity of 1,2-Dimethylhydrazine in in vivo Medium-Term Liver Initiation Assay Ssytem using 4-Week-Old Rats without Hepatocellular Proliferative Stimuli during the Test Chemical Treatment Period

Quantification of cytochrome 2E1 in human liver microsomes using a validated indirect ELISA

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