Cytochromes P450 (CYP, P450) are haem-containing enzymes that catalyse the oxidative metabolism of a multitude of organic compounds. Members of the CYP1-CYP4 families are best known for their key role in drug metabolism, but several drug-metabolising P450s also participate in the metabolism and synthesis of endogenous compounds. Thus, interactions between exogenous and endogenous metabolic P450 pathways are of major interest, as they can alter physiological functions, as well as the pharmacological outcome of drugs that cross the blood-brain barrier. A novel potential endogenous substrate for brain P450s is the tryptophan derivative, indole. In humans, the gut flora can produce significant amounts of indole from the tryptophan ingested with the diet, which are then absorbed through the intestinal wall. Previous studies have shown that several human P450s in CYP1 and CYP2 families can catalyse indole oxidation to isatin, a neuromodulator that exerts anxiogenic effects. Some of the most active P450s, namely CYP2A6, CYP2D6 and CYP2E1, are also known to metabolise, and be regulated by, alcohol and cigarette smoking. This thesis explores the hypotheses that P450s responsible for isatin production are present in regions of the brain relevant to isatin action and that the production of isatin can be affected by interactions with cigarette smoking and alcohol.
To achieve this objective, it was first necessary to characterise the activity of three poorly-characterised P450s, namely CYP2S1, CYP2U1 and CYP2W1, towards isatin production, to extend previous studies on human P450s. Limited knowledge currently exists on their tissue expression and catalytic activities. It was found that all three enzymes were able to catalyse indole oxidation to isatin and oxindole. In particular, incubations with CYP2U1, and with CYP2W1 to a lesser extent, showed the highest levels of metabolite formation. In addition, recombinant expression systems were established for CYP2J2 and CYP4X1. The five proteins were then purified to raise the antibodies needed in chapter four.
The second part of this thesis investigated the expression of several indole-metabolising P450s in the human brain from alcoholics, smokers and drug-free controls. Two brain areas involved both in drug addiction and isatin action are prefrontal cortex (PFC) and amygdala (AMG). However, only a single P450 from families CYP1-CYP4 (namely CYP4X1) has been detected in human amygdala, and no investigations have been performed on P450 transcript expression in brain of alcoholics and smokers.
The studies described in chapter three examined mRNA expression of fifteen P450s, their redox partners, three ABC transporters and four related transcription factors in PFC and AMG of alcoholics, smokers and drug-free controls by quantitative reverse-transcription PCR (qRT-PCR). It was found that CYP1A1, CYP1B1, CYP2B6, CYP2C18, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, CYP4X1, CYP46, cytochrome P450 reductase, Adrenodoxin (Adx), ABCB1, ABCG2, ABCA1, Aryl hydrocarbon Receptor and Peroxisome-Proliferator Activated Receptor α were expressed at levels sufficient for quantification, in both brain areas. CYP2A6, CYP2C19, Adrenodoxin reductase and the transcription factors Pregnane X Receptor and Constitutive Androstane Receptor were also detected in the majority of samples, but were below the limit of quantification, whereas CYP1A2 and CYP2W1 were not detected. Few significant differences were observed between any subject group. Higher levels of Adx mRNA in alcoholics and a trend towards induction of CYP1A1 and CYP1B1 in smokers were observed.
This work was extended in chapter four by an exploration of the expression of selected P450 proteins in the human PFC and AMG. To date, only a few drug-metabolising P450s, namely CYP2B6, CYP2D6 and CYP2E1, have been previously found induced in the alcoholic and smoker human brain at the protein level. However, it is still unclear whether induction occurs transcriptionally or post-transcriptionally, as no study has investigated both protein and transcript expression in the same alcoholic and smoker samples. The experiments reported in chapter four of this thesis examine protein expression of CYP2B6, CYP2E1, CYP2J2, CYP2S1, CYP2U1 and CYP2W1 in a subset of the samples analysed in chapter three by qRT-PCR. No expression of CYP2B6, CYP2J2, CYP2S1 and CYP2W1 was observed, whereas CYP2E1 and CYP2U1 were expressed in every sample. Additionally, protein levels of both P450s were elevated in alcoholics, and CYP2U1 expression was also higher in the brain of smokers. In particular, CYP2E1 induction by alcohol was more evident in AMG, whereas CYP2U1 showed significant induction by either drug only in PFC. Given the lack of variation in CYP2E1 and CYP2U1 transcript expression between case groups, as analysed by qRT-PCR in the same samples, these findings suggest a post-transcriptional regulatory mechanism, potentially through enzyme stabilisation, for both CYP2E1 and CYP2U1.
In summary, the work in this thesis demonstrates the activity of three poorly-characterised P450s in the oxidation of indole to isatin, and the expression of several drug- and indole- metabolising P450s in two brain areas key to isatin activity and drug addiction. In addition, it shows that CYP2E1 and CYP2U1 proteins are up-regulated, most likely at the post-transcriptional level, by drinking and smoking in human AMG and PFC. As CYP2E1 is one of the forms most active in the metabolism of indole to isatin, and the highest induction of CYP2E1 was observed in AMG, a brain area highly relevant to both anxiety and isatin action, these findings make CYP2E1 a prime candidate for a physiological role in this pathway. In particular, if induction by alcohol in the brain occurs through substrate stabilisation of CYP2E1, this would likely result in the inhibition of other metabolic reactions catalysed by this enzyme, including indole metabolism. These results lay the foundation for further studies exploring these hypotheses directly, which might reveal possibilities for new therapeutic strategies against anxiety disorders, through the development of novel P450 inhibitors designed to block isatin production in the brain.