Preparation and Characterisation of S9 Fractions

Hubbard, Susan A.; Brooks, T.M.; Gonzalez, L. P.; Bridges, James W.

doi:10.1007/978-1-349-07901-8_50

Cited by 12 publications

(9 citation statements)

References 20 publications

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“…It is recommended that in vitro systems for genotoxicity screening include the metabolic activation procedure that depends on the use of the rodent liver S9 fraction, which is a postmitochondrial supernatant consisting of both cytosol and microsomes [37,47,59]. The S9 fraction…”

Section: Assessment Of the Effects Of The S9 Mixture Used For Procarcinogen Metabolic Activation On Gfp Reactivation In Hela Ti Cellsmentioning

confidence: 99%

HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes

Maksimova¹,

Shalginskikh

Vlasova³

et al. 2021

PLoS ONE

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Chemicals reactivating epigenetically silenced genes target diverse classes of enzymes, including DNMTs, HDACs, HMTs and BET protein family members. They can strongly influence the expression of genes and endogenous retroviral elements with concomitant dsRNA synthesis and massive transcription of LTRs. Chemicals reactivating gene expression may cause both beneficial effects in cancer cells and may be hazardous by promoting carcinogenesis. Among chemicals used in medicine and commerce, only a small fraction has been studied with respect to their influence on epigenetic silencing. Screening of chemicals reactivating silent genes requires adequate systems mimicking whole-genome processes. We used a HeLa TSA-inducible cell population (HeLa TI cells) obtained by retroviral infection of a GFP-containing vector followed by several rounds of cell sorting for screening purposes. Previously, the details of GFP epigenetic silencing in HeLa TI cells were thoroughly described. Herein, we show that the epigenetically repressed gene GFP is reactivated by 15 agents, including HDAC inhibitors–vorinostat, sodium butyrate, valproic acid, depsipeptide, pomiferin, and entinostat; DNMT inhibitors–decitabine, 5-azacytidine, RG108; HMT inhibitors–UNC0638, BIX01294, DZNep; a chromatin remodeler–curaxin CBL0137; and BET inhibitors–JQ-1 and JQ-35. We demonstrate that combinations of epigenetic modulators caused a significant increase in cell number with reactivated GFP compared to the individual effects of each agent. HeLa TI cells are competent to metabolize xenobiotics and possess constitutively expressed and inducible cytochrome P450 mono-oxygenases involved in xenobiotic biotransformation. Thus, HeLa TI cells may be used as an adequate test system for the extensive screening of chemicals, including those that must be metabolically activated. Studying the additional metabolic activation of xenobiotics, we surprisingly found that the rat liver S9 fraction, which has been widely used for xenobiotic activation in genotoxicity tests, reactivated epigenetically silenced genes. Applying the HeLa TI system, we show that N-nitrosodiphenylamine and N-nitrosodimethylamine reactivate epigenetically silenced genes, probably by affecting DNA methylation.

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Section: Assessment Of the Effects Of The S9 Mixture Used For Procarcinogen Metabolic Activation On Gfp Reactivation In Hela Ti Cellsmentioning

confidence: 99%

HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes

Maksimova¹,

Shalginskikh

Vlasova³

et al. 2021

PLoS ONE

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show abstract

“…In general, JBC 1847 was found to be a non-mutagenic compound with MR values of < 2. However, the MR for S. typhimurium TA1537 was above 2 at a concentration of 4 μg/well in the presence and absence of the metabolic activation system S9 ( Hubbard et al, 1985 ; Table 6 ).…”

Section: Resultsmentioning

confidence: 99%

“…In the present study, S9 was derived from rat liver, and notably, previous studies have shown that the outcome of the Ames test may vary according to whether S9 is prepared from rat or human liver extract (Hakura et al, 2005). The S9 system is applied because many xenobiotics are only mutagenic after being metabolized, e.g., by cytochrome P450 enzymes (Hubbard et al, 1985). In fact, many liver and cutaneous enzymes involved in metabolism are the same.…”

Section: Discussionmentioning

confidence: 99%

A Novel Promazine Derivative Shows High in vitro and in vivo Antimicrobial Activity Against Staphylococcus aureus

et al. 2020

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The emergence of multidrug-resistant bacteria constitutes a significant public health issue worldwide. Consequently, there is an urgent clinical need for novel treatment solutions. It has been shown in vitro that phenothiazines can act as adjuvants to antibiotics whereby the minimum inhibitory concentration (MIC) of the antibiotic is decreased. However, phenothiazines do not perform well in vivo, most likely because they can permeate the blood-brain (BBB) barrier and cause severe side-effects to the central nervous system. Therefore, the aim of this study was to synthesize a promazine derivate that would not cross the BBB but retain its properties as antimicrobial helper compound. Surprisingly, in vitro studies showed that the novel compound, JBC 1847 exhibited highly increased antimicrobial activity against eight Gram-positive pathogens (MIC, 0.5-2 mg/L), whereas a disc diffusion assay indicated that the properties as an adjuvant were lost. JBC 1847 showed significant (P < 0.0001) activity against a Staphylococcus aureus strain compared with the vehicle, in an in vivo wound infection model. However, both in vitro and in silico analyses showed that JBC 1847 possesses strong affinity for human plasma proteins and an Ames test showed that generally, it is a non-mutagenic compound. Finally, in silico predictions suggested that the compound was not prone to pass the BBB and had a suitable permeability to the skin. In conclusion, JBC 1847 is therefore suggested to hold potential as a novel topical agent for the clinical treatment of S. aureus skin and soft tissue infections, but pharmacokinetics and pharmacodynamics need to be further investigated.

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“…There is an opinion that enzymes of microsomal fractions show instability under storage conditions at -20°C and above. For example, some denaturing agents, such as proteases, are active at -20°C (Hubbard et al, 1985). Therefore, the preparation №2 was prepared in a manner similar to that used for preparation №1, except that the finished product was stored at -20°C.…”

Section: Optimization Of the Protocol For Obtaining The Hepatic S-9 Fmentioning

confidence: 99%

Determination of the Mutagenicity of 2-aminoanthracene Using Chicken Hepatic S-9 Fraction

Chistyakov¹,

Усатов²,

Klimenko³

et al. 2018

OnLine Journal of Biological Sciences

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The abundance of potential genotoxins makes it necessary to improve microbial test systems that allow to identify these substances quickly. At the same time, the methodology of metabolic activation of promutagens in vitro is very important. Microsomal preparations of rodents, which are generally used for that, have significant disadvantages associated with the potential health risks of the inducers of the metabolism of microsomal cytochromes. For the metabolic activation of promutagens, we have developed a protocol of microsomal homogenate fraction (S-9) preparation. We propose to complement the set of methods for quality control of feeds for valuable and rare chicken breeds with the Ames test (Salmonella/microsome) using the activated chicken hepatic S-9 homogenate fraction.

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Preparation and Characterisation of S9 Fractions

Cited by 12 publications

References 20 publications

HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes

HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes

A Novel Promazine Derivative Shows High in vitro and in vivo Antimicrobial Activity Against Staphylococcus aureus

Determination of the Mutagenicity of 2-aminoanthracene Using Chicken Hepatic S-9 Fraction

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