Marine Ecotoxicology, F. Regoli

PHASE I

Phase I is carried out by Cytochrome P-450: a multi-enzymatic family localized on the inner side of the

membrane of the smooth endoplasmic reticulum. A characteristic of this system is its complexity, due to the

synergistic action of many enzymes, activated by many signals.

Cp450 is involved in the catalysation of transformation reactions, in many cases oxidations. It contains Iron,

in the form of an heme group; it also requires the presence of a chain of electron transporters and oxygenases

linked to this system, to catalyse the oxidation reaction. Some enzymes included in this family use the same

substrate, others use similar compounds, other enzymes catalyse the oxidation of completely different

compounds. All this ultrastructure is related to guarantee a very simple chemical reaction.

These kinds of reactions in many cases increase the toxicity of certain xenobiotics. This system is regulated

and highly inducible; depending on the levels at which the system is working, we can understand to which

contaminant the organism has been exposed. The induction of cp450 is regulated at the gene level: the first

mechanism of induction of this system is based on the enhanced of gene translation.

This system is regulated and expressed by highly conserved sequences. The real original function of this

system is to metabolize endogenous substrates, in particular:

 to synthetize and degrade steroids, including cholesterol, androgens and oestrogens.

 Fatty acids and derivatives, like arachidonic and lauric acid.

 some plants developed mechanisms to avoid grazing by producing metabolites through this system.

A secondary function of this system is the metabolization of xenobiotic compounds. The classes of

xenobiotics that can be transformed by Cp450 are:

 several kinds of drugs or pharmaceutical compounds (recently becoming of environmental concern,

since they were actually found at some level in marine organisms)

 toxic substances like PAHs, pesticides, insecticides, organo-chlorinated compounds.

These chemicals have structural characteristics that are recognised by this system and can induce its

activation.

Normally, an enzymatic family of Cp450 is identified with an acronym, which is CYP+number/letter. The

number usually indicate the biological function of the enzymes belonging to that family. We arrive as far as

CYP71.

CYP1, 2 and 3 are the more interesting to us, because they’re more involved in the transformation of chemical

pollutant; CYP19 is involved in the synthesis of sexual hormones and it can be modulated by some of these

pollutants. The functions and biological roles of these families are numerous and diverse.

These systems are inducible and induction is mostly transcriptional; anyway, there are other levels on which

induction can act, for example the transformation of the conformational structure of proteins, which can

deeply change their activity. Cp450 is induced because there is something happening at the gene level.

Generally speaking, enzymes of the Cp450 are involved in the catalysis of rather simple, but often

fundamental reactions; for example:

 Demethylation

 Testosterone and oestradiol have a totally different function, but chemically speaking these

compounds are produced by the same enzymatic pathway, involving CYP19 (also called Aromatase);

the reaction is the aromatisation of testosterone to transform it into oestradiol. If this system is not

working in a female organism, it tends to accumulate testosterone, leading to masculinization.

 Epoxidation of arachidonic acid into epoxyeicosatrienoic acid; these molecules are extremely

reactive

 Hydroxylation of a tetrachloro-biphenyl (TCB); this molecule has 4 chlorine atoms and it’s an organic

xenobiotic. An organism accumulating this compound needs to excrete it.

Phase I, chemically speaking, is very simple: the original compound is hydroxylated. From a biological point

of view, on the other hand, this reaction is essential.

Mechanism of Phase I

The aim of the reaction is to transform the xenobiotic, R, into ROH.

1. The first step is that the xenobiotic is bound to the Cp450, localized on the membrane of the SER;

the SER’s function is to synthetize all those compounds that serve as endogenous substrates for any

cellular reaction. Here, lipophilic compounds can be metabolized, in order to be transferred into the

cytosol.

2. The xenobiotic is bound to the enzyme, which is an Iron containing protein; initially, the Iron is in its

3+

oxidized form, Fe . For the proper functioning of the system, the enzyme needs a chain of electron

→

3+

transporters: NADPH-Cp450 reductase. This system reduces Cp450 at the level of the Iron: Fe

2+

Fe .

When Fe is in its reduced form, it can interact with a molecule of Oxygen. With the transfer of the first

electron, Oxygen is bound into the structure of Cp450.

3. A second electron is used for a particular reaction: the same molecule of Oxygen is used to oxidize

3+

two different substrates: the Cp450 Iron, which then returns to its oxidized state (Fe ) and our

xenobiotic. This system is also called Mixed Function Oxygenase.

4. The Cp450 is then ready to perform another reaction.

Total Balance: →

+ +

RH + 2NADPH + O + 2H ROH + 2NADP + H O

2 2

The main difference between R and ROH is that the latter is more water-soluble; in some cases, it’s so

Hydrophilic that it can be excreted without further transformations, while in other cases, a Phase II is

required.

Often, metabolites produced during Phase I are highly reactive. In some cases, they’re so reactive that they

form stable adducts to DNA; the Cp450 system was not evolved by the cell for the toxification of PAHs, so

the result of their metabolism is not ideal. This is the reason why many organic xenobiotics are carcinogenic

compounds.

Different Furans, even when they’re similar from a chemical point of view, have different capability to induce

the expression of genes coding for Cp450. The PAH that would be possibly transformed, before the

transformation is able to increase the capability of the cell to catalyse the Hydroxylation reaction.

The xenobiotic enters within the cell without any energy consumption, thanks to its lipophilic properties.

The molecule Is recognized by the Cytochrome receptor: the Ah (Aromatic Hydrocarbon) transcription factor,

described for the vertebrates, but not for invertebrates. It is normally present in an inactive form, because it

is bound to heath-shock proteins.

At this stage, the planarity of the molecule is extremely important, because it influences the efficiency of the

interaction with the receptor: the inducibility of Cp450 is strictly dependent on the planarity of the

xenobiotic.

Once the molecule reacts with the receptor, this system is activated:

1. The heath-shock protein is released

2. The AhR complex is transferred by a nuclear translocator called ARNT into the nucleus

3. In the nucleus, the complex arrives onto a DNA region, where it finds and interacts with specific

responsive elements (Dioxine Responsive Elements).

These responsive elements are regulatory regions of genes, which modulate their expression. The toxicity of

PAHs is mediated by this mechanism, the interaction with regulatory regions of the genes.

This interaction induces the gene of Cp450 and the cell enhances its capability to transform the xenobiotics.

The complexity of the system is related to the fact that the Dioxine Responsive Elements are located not only

in the regulatory regions of the genes involved in this biotransformation; other genes have these DREs. This

gene induction is responsible not only of the induction of the Cp450 system, but also of the activation or

inhibition of many other cellular pathways, related, for example, to apoptosis, cell cycle, cell proliferation,

→

and others unknown PLEIOTROPIC RESPONSES.

Measure of EROD activity, which is the catalytic activity responsible for the hydroxylation reaction induced

by the xenobiotics; in fish exposed to different compounds in different doses:

 Aroclor 1254 appears to be inducing the EROD activity, meaning that Aroclor reacts with the Ah

receptor and induces gene expression of Cp450, synthesis of proteins and enhancing of the catalytic

reaction of hydroxylation.

 BNF (β-Naphto Flavon) shows a similar behaviour.

 B[a]P (Benzo a Pyrene) shows an even higher capability of inducing EROD activity, because it’s more

planar.

 TCDD (2,3,7,8-Tetrachlorodibenzodioxin) has the highest capability of inducing EROD activity, always

because it has a much higher chemical affinity with the receptor. It’s considered the model

compound to study the induction of Pc450.

This shows a direct correlation between the level of affinity of a xenobiotic for the Ah receptor and its

planarity. When I find an organism, which has high levels of EROD activity, it could be exposed to high

amounts of xenobiotics with a relatively low affinity for the receptor, or to little amounts of xenobiotics with

high affinity for the receptor.

From a practical point of view, another important factor to consider is how long the gene expression is

maintained after contamination. The maintenance of a stimulus depends on the molecules and on the

possibility that the hydrolysed compound would be excreted.

Time of the gene induction in fish (mRNA) which

have been exposed on a single dose of 2

different xenobiotics:

 BNF is rapidly metabolized – after the

contamination, the activity of gene

expression rapidly returns to basal

levels, because the BNF is efficiently

excreted.

 TCDF is slowly metabolized – it’s not

easily excreted, if not at all, so the gene

expression continues to show high

levels after some time, with a constant

transcriptional induction of the system.

It’s important, because when the induction of Cp450 is measured, once highlighted that the organisms have

been exposed to specific classes of chemicals, it is also important to understand when the exposure

happened and if it’s still happening. The expression of mRNA is far less durable than the protein activity:

proteins will remain in the cell longer than the mRNA.

If we compare vertebrates and invertebrates, in terms of biotransformation systems, we can see that

vertebrates show a rapid induction of Cp450 by PAHs; PAHs are also rapidly metabolized. This means that

PAHs, once hydrolysed, can be excreted.

Halogenated Aromatic Hydrocarbons, on the other hand, strongly induce Cp450, but are metabolized very

slowly or not at all. Gene expressions are induced, proteins are produced, the catalytic activity is enhanced,

but the metabolites obtained cannot be further transformed and excreted.

In invertebrates, this system is much less important. The metabolic rate of organic contaminants is much

lower. Compared to fish, invertebrates accumulate much more PAHs, because the xenobiotics cannot be

efficiently transformed in more water-soluble compounds.

Accumulation results from the balance between what enters in the cell, what is metabolized a