Introduction

Human impact on the environment presents an ongoing problem; with approximately 85,000

known chemicals being used today, we need to assure the safety of their use is assessed and

validated1. Various Cytochromes P450 (CYPs) are capable of metabolizing xenobiotic substances,

including potential environmental toxins. Selecting and expressing CYPs from model organisms that

are orthologous to the major drug metabolizing CYPs found in humans should allow for comparison

of their functions and assessment of their capacity in drug detoxification . Environmental

bio-indicator species (EBS) e.g. Danio rerio (Zebrafish) or Oncorhynchus mykiss (Rainbow Trout)

are of particular interest as they are often used as model species for monitoring the environment and

have similar biological pathways to other species2. By establishing the relationship between selected

environmental toxins and the products formed by action of EBS CYPs orthologous to human drug

metabolizing CYPs, I will ascertain the range of substrates, discover if the metabolites are similar to

their human counterparts, and establish if metabolites are potentially toxic and/or inhibit or induce

orthologue CYP activity.

Figure 1; Examples of important environmental toxins. These toxins are currently of concern to environmental agencies and they are known to be

found in the water table. These toxins were shown to be toxic to most aquatic organisms and also pose a threat to other species.

Cytochromes P450

CYPs are found in eukaryotes, numerous prokaryotes and also in the archaea. CYPs in the

human hepatic system have key roles in Phase I drug metabolism. They mediate diverse

(mostly oxidative) reactions on a large range of substrates. They are responsible for the

biotransformation of xenobiotics and ~75% of pharmaceuticals are metabolised by CYPs4.

During Phase I metabolism the biotransformation of the compound occurs, often

involving the addition of an oxygen atom (e.g. as a hydroxyl group) by a CYP enzyme,

resulting in a more hydrophilic product. However, CYP-dependent oxidative metabolism

can also result in other outcomes, including e.g. demethylation/dealkylation,

sulfoxidation, dehydration and epoxidation, as well as more “exotic” transformations

including isomerization, decarboxylation and C-C bond formation5. CYPs thus have

diverse catalytic activities and have been described as ‘Nature’s most versatile catalyst’5.

Environmentally Sensitive Species and their CYPs

Several species were selected as being of particular interest, since they are often used as model

organisms in research projects, and have also been used as bio-indicator species for environment

toxicology. These species play an important role in monitoring environmental pollution as they

demonstrate sensitivity to changes in aquatic environments. The use of biochemical markers such

as cytochromes P450 helps determine the mechanism of toxicity of a pollutant, providing insights

into the effects of the pollutant and its potential metabolites on the organism7. There is an

increasing emphasis on understanding the structure and function of key enzymes that are conserved

across species. Selecting key species that demonstrate similarity in relevant biological pathways to

other species (e.g. drug metabolism) can expedite the use of these non-traditional species as

toxicological models. In this project, I will examine species possessing orthologues of human CYP

genes, and establish if these orthologues may have conserved functions in metabolising xenobiotics

and/or can make non-human type metabolites that cause toxicity to environmentally sensitive

organisms8.

Conclusions and Ongoing Work

Cytochrome P450s have an important and evolving role in both pharmaceutical and

toxicological research. New and interesting data have shown how CYPs are involved in

adverse drug reactions, the toxicity of natural and man-made compounds and the activation

of toxins. How CYPs mediate these reactions is of great importance for a range of industries

and agencies. Microsomal CYPs are difficult to study but will provide a greater insight into

the mechanisms involved in xenobiotic transformation in our target species. To produce ESS

P450 enzymes, in vitro studies will be done using E. coli as the expression host. Selected

CYPs from the species discussed will be cloned into appropriate vectors to express the

proteins. Several CYP genes have already been identified, synthesized and cloned into

vectors. These CYP genes were synthesized to include a restriction site enabling (as required)

removal of the N-terminal transmembrane helix and were codon optimized for their host

E. coli (see Table 1). Preliminary results have revealed that five out of six initial clones show

CYP/CPR expression.

Cytochrome

p450 Species Class

Orthologous CYP

from Homo sapiens

% AA

Identity

CYP3A126 Pimephales promelas Teleost—Cyprinid CYP3A4 and CYP3A5 56.6/54.1

CYP3A27

Oncorhynchus mykiss

CYP3A4 and CYP3A5 53.3/52.3

Teleost—Salmonid

CYP3A45 52.9/52.7

CYP1A1

CYP1A2

51.6

CYP1A2 52.2

CYP1A3 52.4

CYP2K5 CYP2C9

44.2

CYP2M1 44.9

CPR CPR 72.4

CYP3A65

Danio rerio Teleost—Cyprinid

CYP3A4 and CYP3A5 54.1/51.8

CYP3C1-4 48.6/47.5

CYP1A CYP1A2 52.9

CPR CPR 73.9

Figure 4; The catalytic cycle of typical cytochromes P450. The stars indicate likely

rate-limiting steps in the activity of CYPs—which vary depending on the CYP. These include

the first electron reduction of P450 FeIII to FeII, and the dissociation of the product. The cy-

cle begins with substrate (RH) binding to displace the H2O ligand in the distal position)6.

Figure 3; Diagram showing the

heme binding pocket of CYP3A4

bound to ketoconazole. Ketocona-

zole is shown in turquoise, the heme is

shown in brick red and the secondary

protein structure are represented in

silver grey (PDB ID 2VoM). Three

molecules of ketoconazole are bound

in the CYP3A4 active site.

Figure 2. Diagram showing the

heme binding pocket of CYP3A4

bound to erythromycin. Erythro-

mycin is shown in turquoise, the heme

is shown in purple and the protein

secondary structure is represented in

green (PDB ID 2J0D)3.

Figure 5; Schematic of electron transfer reactions in microsomal and bacterial P450 redox systems. Panel A shows both a P450 and its cytochrome

P450 reductase (CPR) redox partner, both associated with the ER membrane through a N-terminal transmembrane anchor region. Electron transfer occurs from

NADPH through FAD and FMN cofactors to the P450 heme iron, ultimately leading to the activation of O2 bound to the heme iron and to its scission and the inser-

tion of an oxygen atom into the substrate (RH) to form a hydroxylated product (ROH). Panel B shows a similar reaction scheme for a soluble, bacterial P450-CPR

fusion enzyme (e.g. P450 BM3 [CYP102A1]). Neither of the domains of this enzyme have membrane anchor regions. (Adapted from Gueguen et al, 2006 )4.

Figure 6; Phylogenetic tree showing the relationship between CYPs in Homo sapiens and in three fish species of interest; The fish selected are

Pimephales promelas, Oncorhynchus mykiss and Danio rerio. This tree demonstrates the phylogenetic relationships between the species, and the orthologous rela-

tionships between the CYP enzymes. The functions of these CYPs may be largely conserved as the species have evolved and diverged.

Table 1; Orthologues identified by bioinformatics. Those genes shown in purple have been cloned and are undergoing expression trials.

References

1. Erikson, B. E., How many chemicals are in use today? EPA struggles to keep it's chemical inventory up

to date. Chem. Eng. News February 2017, 95 (9), 23-24

2. Siroka, Z.; Drastichova, J., Biochemical markers of aquatic environment contamination - Cytochrome

P450 in fish. A review. Acta Veterinaria Brno 2004, 73 (1), 123-132

3. Ekroos, M.; Sjogren, T., Structural basis for ligand promiscuity in cytochrome P450 3A4. Proc. Natl.

Acad. Sci. U. S. A. 2006, 103 (37), 13682-13687

4. Gueguen, Y.; Mouzat, K.; Ferrari, L.; Tissandie, E.; Lobaccaro, J. M. A.; Batt, A. M.; Paquet, F.; Voisin,

P.; Aigueperse, J.; Gourmelon, P.; Souidi, M., Cytochromes P450: xenobiotic metabolism, regulation

and clinical importance. Annales De Biologie Clinique 2006, 64 (6), 535-548

5. Coon, M. J., Cytochrome P450: nature's most versatile biological catalyst. Annu. Rev. Pharmacol. Tox-

icol. 2005, 45, 1-25

6. Corsini, A.; Bortolini, M., Drug-induced liver injury: the role of drug metabolism and transport. J.

Clin. Pharmacol. 2013, 53 (5), 463-474

7. Munro, A. W.; Girvan, H. M.; Mason, A. E.; Dunford, A. J.; McLean, K. J. What makes a P450 tick?

Trends Biochem. Sci. 2013, 38 (3), 140-150.

8. Siroka, Z.; Drastichova, J., Biochemical markers of aquatic environment contamination - Cytochrome

P450 in fish. A review. Acta Veterinaria Brno 2004, 73 (1), 123-132.

Emily Fox. Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, 131 Princess Street, Manchester M1 7DN