Hepatic drug metabolizing profile of Flinders Sensitive Line rat model of depression

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Abstract

The Flinders Sensitive Line (FSL) rat model of depression exhibits some behavioral, neurochemical, and pharmacological features that have been reported in depressed patients and has been very effective in screening antidepressants. Major factor that determines the effectiveness and toxicity of a drug is the drug metabolizing capacity of the liver. Therefore, in order to discriminate possible differentiation in the hepatic drug metabolism between FSL rats and Sprague–Dawley (SD) controls, their hepatic metabolic profile was investigated in this study. The data showed decreased glutathione (GSH) content and glutathione S-transferase (GST) activity and lower expression of certain major CYP enzymes, including the CYP2B1, CYP2C11 and CYP2D1 in FSL rats compared to SD controls. In contrast, p-nitrophenol hydroxylase (PNP), 7-ethoxyresorufin-O-dealkylase (EROD) and 16α-testosterone hydroxylase activities were higher in FSL rats. Interestingly, the wide spread environmental pollutant benzo(α)pyrene (B(α)P) induced CYP1A1, CYP1A2, CYP2B1/2 and ALDH3c at a lesser extend in FSL than in SD rats, whereas the antidepressant mirtazapine (MIRT) up-regulated CYP1A1/2, CYP2C11, CYP2D1, CYP2E1 and CYP3A1/2, mainly, in FSL rats. The drug also further increased ALDH3c whereas suppressed GSH content in B(α)P-exposed FSL rats. In conclusion, several key enzymes of the hepatic biotransformation machinery are differentially expressed in FSL than in SD rats, a condition that may influence the outcome of drug therapy. The MIRT-induced up-regulation of several drug-metabolizing enzymes indicates the critical role of antidepressant treatment that should be always taken into account in the designing of treatment and interpretation of insufficient pharmacotherapy or drug toxicity.

Introduction

Previous studies reported that depressed individuals show increased vulnerability in developing several pathologies including immune diseases and cancer, whereas effective psychotherapeutic treatment of depression affected the course of cancer (Hilakivi-Clarke et al., 1993, Spiegel, 1996, Sheibani-Rad and Velanovich, 2006, De Jonge et al., 2006, Kawajiri, 1999). It is of interest to note that stress, which is tightly connected to the pathophysiology of depression (Johnson et al., 1996, Chaouloff, 2000, Chaouloff et al., 2007), stimulated the development of cancers (Hilakivi-Clarke et al., 1993, Fox, 1995). Interestingly, the tumor growth was increased after chronic stress amongst rats that had developed depression-like behavior (Sklar and Anisman, 1981).

Most of the drugs are metabolized by the so-called drug-metabolizing enzymes, including phase I (oxidative enzymes) and phase II (conjugating enzymes). The cytochromes P450 (CYP) are amongst the most important phase I enzymes and many drugs, carcinogens and toxicants are known to be metabolically activated by certain cytochrome P450 isoforms, suggesting a role for P450s in drug efficacy, toxicity and cancer (Rodriguez-Antona and Ingelman-Sundberg, 2006, Gonzalez and Yu, 2006). On the other hand, some of the phase II enzymes, such as the glutathione transferases and its coenzyme, glutathione, play an important role in protecting the cells against toxic and carcinogenic metabolites produced by P450s (Foliot and Beaune, 1994). It should be noted also that a decisive risk factor for getting cancer, is the relative levels of activating versus inactivating enzymes in the cells, in addition to the levels of a carcinogen an individual is exposed to (Kawajiri et al., 1993). Members of four CYP families primarily metabolize xenobiotics including families CYP1 through CYP4. The CYP1 family is the most notable for carcinogen and toxicant metabolism, whereas CYP2 and CYP3 families metabolize numerous clinically used drugs and other compounds, ultimately resulting, after phase II metabolism, in most cases, in more stable and hydrophilic derivatives that are readily eliminated (Gonzalez and Yu, 2006).

Polycyclic aromatic hydrocarbons (PAHs), widely spread environmental pollutants, present in heavily polluted air of industrial and urban areas, originate from various sources, such as vehicle exhaust emissions, refuse burning, cooking and cigarette smoke (Mastral et al., 2001, Miller and Ramos, 2001). Benzo(α)pyrene (B(α)P), the prototype PAH, and related substances have a dual role, acting as inducers of CYP1A1, CYP1A2 and CYP1B1 genes and as pre-carcinogenic substrates for the corresponding enzymes, and they are considered as particularly important toxicants (Pelkonen and Nebert, 1982, Nebert, 1989, Gonzalez, 1988, Ioannides and Parke, 1990, Kawajiri et al., 1993, Baumann and Harshbarger, 1995, Mumford et al., 1995, Huntley et al., 1993, Whitlock et al., 1996, Nebert et al., 2000, Wu et al., 2002, Wang et al., 2007).

We have previously shown using experimental animal models, that stress modulates the expression of several drug and carcinogen metabolizing enzymes (Konstandi et al., 2004, Konstandi et al., 2005). As stress, in many cases, underlies depression (Johnson et al., 1996, Chaouloff, 2000, Chaouloff et al., 2007), this finding could imply that the link between depression and increased morbidity and cancer incidence may be due, at least in part, to modified hepatic drug and carcinogen metabolism.

Several animal models of depression with different values in paralleling different parameters of human major depression have been used in psychopharmacological research (Overstreet et al., 2005). The Flinders Sensitive Line (FSL) rat model, selectively bred from Sprague–Dawley (SD) rats (Lavi-Avnon et al., 2005), is one of a few useful genetic animal models of depression that have shown face, construct and predictive validity, and with a reasonably well-understood neurobiological basis of behavioral differences (Overstreet, 2002, Overstreet et al., 2005) that were improved by antidepressant treatment (Overstreet, 2002). In contrast to the FSL rats, Sprague–Dawley (SD) rats do not express depressive-like behavior or neurochemical abnormalities (Overstreet, 2002).

Given the fact that the efficacy and toxicity of drugs, including antidepressants, is tightly connected to the drug-metabolizing capacity of the liver, the objective of this study was to investigate the hepatic drug metabolizing profile of the Flinders Sensitive Line rats, the experimental model of depression, which is widely used in psychopharmacological research. For this purpose, the expression of the main CYP enzymes involved in the metabolism of the majority of prescribed drugs in depression and other diseases was evaluated in FSL rats and compared to controls. This assessment took place at a constitutive state and after exposure to benzo(α)pyrene (B(α)P), which induces an AhR-regulated gene battery, including isoforms of CYP1A and ALDH (Nebert, 1989, Nebert et al., 2000). The regulatory role of mirtazapine (MIRT), a widely prescribed antidepressant drug, was also evaluated.

Section snippets

Materials and methods

The experimental animals used in this study were adult male FSL (N = 50) rats, 8–9 weeks old, originated from a colony selectively bred from SD rats at the Center for Alcohol Studies at the University of North Carolina. A pilot study indicated that SD rats display similar hepatic drug metabolizing profile with the Flinder's Resistant Line (FRL) rats (Suppl. Fig. 4), which have been used as controls in several previous studies. In this study, adult male SD rats (N = 50) of the same age (Pasteur

Assessment of the depressive state of FSL rats

FSL rats showed a markedly higher immobility score in the forced swim test compared to SD controls [Mean (in s) ± SE; FSL versus SD; 243.8 ± 10.4 vs 115.9 ± 13.5; p < 0.001].

Assessment of constitutive hepatic drug-metabolizing profile: role of mirtazapine

Although, total P-450 content ranged at the same level in SD and FSL rats, the expression of several major drug-metabolizing enzymes was essentially different in the liver of both strains. It is noteworthy that MIRT treatment increased t-P450 content only in SD rats (Fig. 1, p < 0.05).

FSL rats compared to SD controls displayed higher

Discussion

Thorough analysis of hepatic microsomal and cytosolic drug metabolizing systems showed that the animal model of depression, the FSL rats, displays a substantially distinct hepatic drug metabolizing profile as compared with SD controls. In this regard, mainly two findings are of great significance: on one hand, the lower constitutive hepatic CYP1A2, CYP2B1, CYP2D1, GST expressions and GSH content and the elevated PNP, EROD and 16α-testosterone hydroxylase activities in FSL rats compared to SD

Acknowledgment

This study was funded in the framework of “Heraklitos” under FP3 of EU (MIS 88728/2.2/1736/9). We would also like to thank Drs. Mats Hinderstand and Martha Nicolaidou for their invaluable guidance in optimizing the conditions for the HPLC enzyme analyses and Dr. Gihad Albucharali for his assistance in the hormonal determinations.

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