Anthracyclines and their metabolism in human liver microsomes and the participation of the new microsomal carbonyl reductase

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Abstract

Anthracyclines (ANTs) are widely used in the treatment of various forms of cancer. Although their usage contributes to an improvement in life expectancy, it is limited by severe adverse effects—acute and chronic cardiotoxicity. Several enzymes from both AKR and SDR superfamilies have been reported as participants in the reduction of ANTs. Nevertheless all of these are located in the cytosolic compartment. One microsomal reductase has been found to be involved in the metabolism of xenobiotics—11beta-HSD1, but no further information has been reported about its role in the metabolism of ANTs. The aim of this study is to bring new information about the biotransformation of doxorubicin (DOX), daunorubicin (DAUN) and idarubicin (IDA), not only in human liver microsomal fraction, but also by a novel human liver microsomal carbonyl reductase that has been purified by our group. The reduction of ANTs at C-13 position is regarded as the main pathway in the biotransformation of ANTs. However, our experiments with human liver microsomal fraction show different behaviour, especially when the concentration of ANTs in the incubation mixture is increased. Microsomal fraction was incubated with doxorubicin, daunorubicin and idarubicin. DOX was both reduced into doxorubicinol (DOXOL) and hydrolyzed into aglycone DOX and then subsequently reduced. The same behaviour was observed for the metabolism of DAUN and IDA. The activity of hydrolases definitely brings a new look to the entire metabolism of ANTs in microsomal fraction, as formed aglycones undergo reduction and compete for the binding site with the main ANTs. Moreover, as there are two competitive reducing reactions present for all three ANTs, kinetic values of direct reduction and the reduction of aglycone were calculated. These results were compared to previously published data for human liver cytosol. In addition, the participation of the newly determined human liver microsomal carbonyl reductase was studied. No reduction of DOX into DOXOL was detected. Nevertheless, the involvement in reduction of DAUN into DAUNOL as well as IDA into IDAOL was demonstrated. The kinetic values obtained were then compared with data which have already been reported for cytosolic ANTs reductases.

Introduction

Carbonyl reductases constitute a group of enzymes which play a significant role in physiological processes. Not very much is known about their role in the metabolism of xenobiotics, but it is obvious that they are an important part of the phase I biotransformation of xenobiotics. Drugs are usually substrates for multiple biotransformation pathways; however some drugs undergo reduction as their main metabolic route, including reduction of carbonyl group [1]. Carbonyl-reducing enzymes belong to two superfamilies—short-chain dehydrogenases/reductases (SDR) [2] and aldo-keto reductases (AKR) [3]. The majority of the xenobiotic carbonyl reductases known today are cytosolic enzymes belonging either to AKR (e.g. AKR1C1-4, AKR1B10) or SDR (e.g. CBR1). There is limited knowledge about microsomal carbonyl reducing enzymes which contribute to the metabolism of drugs. Only one well-known microsomal carbonyl reductase which plays a role in the metabolism of xenobiotics—11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) has been reported so far [4], [5]. This enzyme belongs to the SDR superfamily, which in contrast to the AKR superfamily includes not only cytosolic but also microsomal carbonyl reducing enzymes (e.g. 17β-HSD7, RDH 11). However, the activity of microsomal enzymes, with the exception of 11β-HSD1, has so far been reported towards endogenous substrates. Moreover, the SDR superfamily also contains several poorly characterized enzymes, with membrane or unknown subcelullar localization [6]. Recently, our workgroup partially purified a new microsomal carbonyl reductase which possesses activity towards the anticancer drug oracin and most likely belongs to the SDR superfamily [7].

The first anthracyclines (ANTs) – daunorubicin (DAUN) and doxorubicin (DOX) – were isolated in the 1960s and are widely used at the present time in the treatment of various forms of cancer. DOX exhibits a broad spectrum of antitumor activity (e.g. in solid tumors as breast or ovarian carcinoma, pediatric cancers, non-Hodgkins lymphoma), whereas DAUN is mainly indicated in the treatment of leukemias [8]. Although ANT chemotherapy has contributed to an improvement in life expectancy, its usage is limited by severe adverse effects – acute and chronic cardiotoxicity – that can even lead to congestive heart failure. Although ANT cardiotoxicity is widely studied and several mechanisms have been suggested, an exact molecular pathogenesis is currently unknown [9]. One of the proposed mechanisms is a toxicity caused by reduced metabolites [10], [11], [12], [13]. To determine adverse effects as well as improvements in anticancer potency, many ANT derivates have been investigated, but only idarubicin (IDA) and epirubicin (EPI) have so far been incorporated into routine clinical practice [9], [14], [15].

Anthracyclines are generally metabolized by the two-electron reduction of a side chain C-13 carbonyl group to corresponding secondary alcohol metabolites (Fig. 1). The reaction itself is mediated by several carbonyl reducing enzymes; it has been reported that DOX biotransformation to doxorubicinol (DOXOL) is catalyzed by two enzymes from the SDR superfamily – CBR1 and CBR3 [16], [17], [18], [19] along with several AKR enzymes – AKR1A1, AKR1B1, AKR1B10, AKR1C3 and AKR1C4 [18], [20], [21]. The reduction of DAUN to daunorubicinol (DAUNOL) is performed by the same SDR enzymes as DOX – CBR1 and CBR3 [16], [17], [22], [23] along with three AKR enzymes – AKR1A1, AKR1B1 and AKR1B10 [20], [22], [24], while results concerning the role of AKR1C2 in DAUN metabolism have proven inconsistent [22], [25]. Moreover, further metabolites, mainly DOX and DOXOL aglycones, have been detected in plasma, but at much lower concentration [15]. It has been reported that DOX is reduced in liver predominantly by CBR1 [18]. However it is still unclear which enzyme is the most important in the biotransformation of both DOX and DAUN in human tissues or tumors. Information about the biotransformation of newer anthracyclines like IDA and EPI is even more scarce; these are also metabolized to C-13 secondary alcohols, but no further knowledge about the participation of particular carbonyl reductases enzymes has been reported so far. Nevertheless it is clear that all currently described carbonyl reductases involved in ANT biotransformation are located in the cytosol, which leaves totally unexplored the area of anthracycline metabolism in microsomal fraction. As a member of membrane-bound reductases, 11β-HSD1 contributes to the biotransformation of several xenobiotics but no results indicating the participation of 11β-HSD1 in the metabolism of ANT has been published yet.

The reduced metabolites DOXOL and DAUNOL are significantly less potent than their parent drugs in terms of inhibiting tumor cell growth in vitro [26]. It also has been reported that carbonyl reducing enzymes are inducible by the presence of ANTs in tumor cell lines. Hence reduction as a detoxification mechanism may contribute to tumor resistance towards DOX or DAUN therapy [26], [27], [28]. In addition, a higher cardiotoxicity of the ANT metabolites have been demonstrated in comparison with their parent drugs, but the exact mechanism is not fully understood, and results concerning the cardiotoxicity of ANTs are generally inconsistent [10], [11], [12]. Idarubicinol (IDAOL), C-13 reduced metabolite of IDA, displays entirely different properties than the metabolites mentioned above. In vitro studies indicate that IDAOL retains good cytotoxic activity comparable with its parent drug. This is thought to be the reason that IDA is five to ten times more potent than DAUN as well as also less cardiotoxic [29]. Similarly to DOX and DAUN, induction of reducing activity towards IDA has been reported for both in vitro and in vivo. However in this case the mechanism leads to the pharmacokinetic self-potentiation of IDA [30]. Thus it is clear that the carbonyl reduction of a selected ANT results in diverse consequences in terms of cytotoxic and adverse effects.

The process of biotransformation of ANTs is far from simple. Besides the main pathway, carbonyl reduction located on C-13, other metabolic pathways are also present and various enzymes are involved in these reactions (Fig. 2).

The complexity and rarity of ANTs metabolism in human cells can be demonstrated by several examples. Some older studies suggested that the formation of reactive oxygen species (ROS) contribute to the anticancer effect of ANTs, but nowadays ROS are oppositely considered to be the cause of cardiotoxicity [9]. Another example is a role of aglycones, which is very insufficiently understood, although it has been reported that aglycones also may be responsible for cardiotoxicity [31] and are probably less cytotoxic to cancer cells than parent drugs [32], [33]. Nevertheless the overall knowledge about cell level of each metabolite is very unsatisfactory and should be researched further.

The participation of particular enzymes in the biotransformation of ANTs has been reported in several publications, but in almost all cases a purified or recombinant form of enzymes was used. Hence the how the whole process takes place on a cellular level has not been clarified. At most some authors have examined the role of human subcellular compartments, but almost all studies have focused completely on cytosol and not on other factors such as microsomal enzymes. The first kinetic parameters for an entire cytosolic fraction were published in a paper in the 1970s [34]. This paper compared the differences of DAUN and DOX reduction caused by cytosol from different tissues and in various mammalian species. Mordente et al. [13] focused on heart cytosol in humans and rabbits to find out if potentially cardiotoxic DOXOL and DAUNOL are formed. A recent publication from Kassner et al. [18] was aimed at particular enzymes in whole cytosol and reported that the most active in the metabolism of DOX is cytosol from the liver and kidney, whereas the lowest clearance was observed in the lung and heart. As indicated above, the situation in the microsomal fraction compared to cytosol is quite unclear. It has not been published whether human microsomal enzymes are involved in the carbonyl reduction or hydrolysis of ANTs. Limited information was mentioned by Watanabe et al. [35], that DOX is metabolized by human liver microsomal enzymes but this activity was expressed only as a decrease in DOX levels, with no information about the formed product.

The aim of this study is to bring new information about the biotransformation of ANTs and in addition to determine whether ANTs are metabolized by human liver microsomal enzymes. All presently known carbonyl-reducing enzymes contributing to the biotransformation of ANTs are cytosolic proteins. Despite the fact that microsomal enzymes are often expressed in lower concentrations than cytosolic ones, it is important to find out whether human microsomal fraction possesses a reductive activity towards ANTs. Recently our workgroup successfully performed a partial purification of the new human liver membrane-bound carbonyl reductase, which, besides microsomal 11β-HSD1, participates in the biotransformation of the anticancer drug oracin [7]. As the typical property of all carbonyl reductases is their wide substrate specificity, it was more than probable that the new microsomal enzyme would also contribute to the biotransformation of other substrates containing carbonyl group. Therefore, in addition to whole human microsomal fraction we also examined the activity of the new microsomal carbonyl reductase towards DOX, DAUN and IDA.

Section snippets

Chemicals

Daunorubicin hydrochloride and idarubicin hydrochloride were obtained from Sigma-Aldrich (Prague, Czech Republic). Doxorubicin hydrochloride, doxorubicinol hydrochloride and daunorubicinol were supplied by TRC (Toronto, Canada). Enzymatic tests were performed by NADP+ and glucose-6-phosphate from Sigma–Aldrich (Prague, Czech Republic) and glucose-6-phosphate dehydrogenase from Roche diagnostics (Mannheim, Germany). Acetonitrile (gradient grade) was obtained from Merck (Prague, Czech Republic).

Participation of microsomal enzymes in biotransformation of ANTs

In this study, the kinetics of ANTs metabolism in human liver microsomal fraction was studied in a substrate concentration range of between 40 and 1300 μM. We are aware that this range is significantly greater than the usual concentration of ANTs present in plasma. But this study is not intended to monitor plasma levels of ANTs. It is closely focused on cell activity, specifically in microsomal fraction and for this purpose, this concentration range is acceptable. Additionally it has been

Conclusions

Results obtained for the biotransformation of ANTs in human liver microsomal fraction indicate that there are two possible pathways for metabolism of ANTs. The first is direct reduction of ANT to its reduced form. The second pathway and most likely of greater importance is hydrolysis of ANT to its aglycone and subsequent reduction to reduced aglycone. Direct hydrolysis of reduced ANT into reduced aglycone has been completely ruled out by experimentation.

Certainly the concentrations used here

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgements

This project was supported by the Grant Agency of Charles University, Grant No. 45508/C/2008, and the grant SVV-2010-261-003. L. Škarydová and H. Štambergová was supported by USPHS NIH grant R13-AA019612 to present this work at the 15th International Meeting on Enzymology and Molecular Biology of Carbonyl Metabolism in Lexington, KY USA. We are grateful to the Transplant Centre (Hradec Kralove) for providing human liver samples.

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