Metabolism and Biotransformation of Pesticides
Biotransformation refers to alteration of a chemical by a biological system. It includes microbial action,
but excludes (for example) photodegradation. Metabolism is often used as a synonym for
biotransformation, but this is not strictly accurate. Metabolism is a somewhat narrower term.
In vertebrates, the liver is the major metabolic organ, as well as the largest internal organ1. Many of the
enzymes present in liver are present, albeit at lower levels, in other organs and tissues: notably in the
gastrointestinal (GI) tract, lungs, skin, and nasal epithelium. In short, any organ that is likely to have
contact with xenobiotics has the metabolic capacity of transforming them. However, most ingested
compounds pass from the GI tract to the liver:
intestinal villi  portal vein  liver  vena cava  heart  general circulation
This means that the liver, with its tremendous metabolic capacity, acts on most ingested substances
before other cells are exposed to them. The major exception is that ingested fats go from the intestines
into the lymph ducts, and are carried to the vena cava by lymph. Thus fats go into the general circulation
without having passed through the liver.
The major biotransforming enzymes of the liver are divided into Phase I and Phase II enzymes. Phase I
enzymes are primarily oxidative, but not exclusively so; some reduction also occurs. Hydrolysis is a
very important Phase II reaction: for example, esterases (the target of insecticidal OPs) are hydrolytic
enzymes, with fatty acid esters as their normal substrates.
Figure 1: Phosphoric acid esters (a, b), Carboxy ester (c), active site of a cholinesterase (d)
The OPs (a, b), are esters of phosphoric acid, and so are analogous to carboxy esters of the general
structure (c). The active site of cholinesterases (d) contains a serine-hydroxyl group which can bind to
carboxy esters, and also to phosphate esters if and only if the P of the OP has a positive charge bind to
the OP. If the OP is P=S, S acts as an electron donor, and P does not have enough positive charge to
provide affinity to the serine OH. If the OP is in the form P=O, O withdraws electrons and allows
binding between the OP and the choline esterase. Following this binding, an epoxide is transiently
1
Actually, skin is the largest organ of the body.
formed and rapidly hydrolyzed, the leaving group is cleaved from the OP (with the addition of water)
and the OP phosphorylates the serine hydroxy-residue.
Thus the essential reaction in OP toxicosis, both in insects and vertebrates, is
P=S  P=O
Only in the P=O form (or the equivalent, see below) will P+ be sufficiently positive to bind to the serine
hydroxyl residue.
The enzymes responsible for catalyzing the P=S  P=O conversion are the cytochromes P-450. These
enzymes, originally called mixed function oxidases or microsomal oxidases are a very large (and still
growing?) family of enzymes first identified in the 1950s by their absorption of light at 450 nm when
carbon monoxide was bubbled through a microsomal suspension. in the early 1960s, Omura and Sato
calculated the extinction coefficient of the cytochromes P-450 (P450s); despite their primitive technique,
the number has never been improved. During the 1960s it became clear that there were at least 2 P450s,
since some chemicals induced a P450 that absorbed light at 448 nm rather than at 450 nm. It was first
thought that each of these 2 enzymes was multifunctional across a wide range of chemicals, but in the
past 40 years it has become clear that there are many P450s, each with a very narrow range of substrates.
They have been divided into families, with a highly complex notation to differentiate the same (or
similar) isozymes occurring in different species, different isozymes occurring in the same species,
and/or to differentiate between a gene and its protein. Moreover, isozymes are grouped by substrate. For
example:
CYP 1A1 designates the gene (capital letters) for the isozyme induced by TCDD2 and related
compounds3 CYP1A1 is one of the isozymes that absorb light at 448 nm.
2
2,3,7,8-tetrachlorodibenzodioxin, often referred to simply as dioxin, an industrial byproduct that is the
most toxic synthetic chemical known.
3
Including coplanar PCBs, dibenzofurans and diphenyl ethers.
CYP 2B1 is the phenobarbital-induced P450. Phenobarbital was one of the first xenobiotics used to
induce (increase the levels of) P450s; CYP2B1 is one of the isozymes that absorb light at 450
nm.
CYP2E1 is induced by ethanol;
Other P450s initiate the conversion of cholesterol to steroids and metabolize estrogen. The CYP 3A
family is induced by phenobarbital, but also by glucocorticoids4.
For our purposes, P450s are the enzymes that make xenobiotics more water soluble, rendering them
more (or less) toxic in the process. For example: CYP 2B is thought to be the isozyme that converts P=S
to P=O.
As shown in Figure 2 (above), P450 mediated activation occurs by way of an epoxide, which rearranges
to form P = O.
Elemental sulfur (S) is released as a result of this reaction. While small quantities of sulfur are not
terribly harmful (and certainly not important relative to the toxicity of the P=O compound), large
amounts of S inhibit P450s, which will slow the conversion of P=S to P=O.
Once formed, the P=O compound is quite reactive, because O withdraws electrons from the P, leaving it
with a relatively positive charge. Hydrolysis of any of the other P-ester bonds occurs quite readily5. It is
also possible for the epoxide to rearrange so that the P=S bond remains, and the leaving group is
cleaved, with the P-O- forming a P-OH at the site of the leaving group. This would result in an inactive
OP – one that no longer has affinity for AChE.
Metcalf and Fukuto showed that the affinity of an OP for AChE was directly correlated with its
hydrolyzability. If X denotes the leaving group of the OP, the general form of the hydrolysis is:
P-O-X  H-O-X + P-O-H
Figure 5 shows this reaction for methyl paraoxon:
Figure 3: Hydrolysis of paraoxon
4
Glucocorticoids (e.g., cortisone) are hormones or intercellular signaling molecules; in particular, they
are part of the vertebrate response to stress; they also mediate inflammatory responses.
5
Note that OPs can also have one or more P-C bonds. These are not readily hydrolyzable and tend to be
quite stable.
Each OP molecule that interacts with AChE (or a nonspecific ChE) destroys one molecule of enzyme.
Note that it is possible for the O-CH3 bond to be cleaved, but this occurs less readily than cleavage of
the p-nitrophenyl group.
Phase II enzymes are conjugation enzymes, which act to increase water solubility, transportability, and
excretion of xenobiotics by hooking them to normal body constituents, such as glutathion 6, UDPglucuronic acid, or a sulfate (SO4) group. Conjugating enzymes, such as glutathion-S-transferase (GST)
catalyze these conjugations., which occur at the P=O bond. In the case of GST, the attachment is to a
sulfhydryl group in glutathion. GST can also pick up the leaving group of the OP (p-nitrophenol, in the
case of paraoxon).
In determining the fate of OPs in the body, and their probable toxicity, it is important to remember that
highly lipid compounds can enter into (and cross through) cell membranes more easily than more polar
molecules. Paraoxon generated by liver P450 is unlikely to cause poisoning, because it is too polar to
get into the nervous system. In fact, upon release from the liver it will probably bind very rapidly to the
(nonspecific) serum cholinesterases, and thus inactivated. Any parathion that is not metabolized in the
liver, however, will not interact with serum esterases, and can cross into the nervous system. (There are
P450s in brain, and presumably also in other nervous system tissue). Therefore, activation of OPs in the
liver probably functions as a detoxifying mechanism, while activation within the nervous system
provides the active molecules that cause neurotoxicity.
Conservative estimates are that P450 levels in the mammalian brain are about 1% of those in liver.
Insects have no blood-brain barrier, and no equivalent to blood vessels. Insect hemolymph is kept
moving throughout the body spaces by a ‘heart’ that keeps hemolymph moving, but not directionally.
One type of OP (among the millions of possible structures) is represented by phorate (figure 6).
Figure 4: phorate
In such compounds, it is also possible to oxidize the S in the leaving group, either to the sulfoxide (S=O)
or the sulfone (O=S=O), a reaction which is not catalyzed by the P450s but by a similar (FAD) enzyme.
If this occurs, the P+ is great enough to allow interaction with AChE even though the P=S bond is
retained. Such OPs are highly reactive. They tend to be used as systemic insecticides: that is, they are
taken up by plants, converted to the active metabolite by plant P450 enzymes, and so poison the plant
parts eaten by the pest insects. They are somewhat less environmentally disruptive, since they do not
threaten nontarget insects as greatly. Systemic insecticides are very useful against sucking insects such
as aphids. One must, of course, time applications carefully so that the insecticide has been eliminated
before the crop is harvested and eaten.
6
Glutathion is a small peptide, 3 amino acids in length; one of the amino acids is glutamate.
Selective toxicity in OPs can be achieved in a number of ways. Malathion is an example of an
insecticide with a carboxyester moiety that is readily cleaved by vertebrate – but not by insect –
carboxyesterases. This provides an excellent safety ration for malathion7. For O-alkyl OPs, the O-ethyl
group is more readily hydrolyzed by insect than by mammalian P450s, while the O-methyl group is
more readily hydrolyzed by mammalian than by insect P450s. Therefore, the methyl analogs of OPs are
to be preferred, and have been selected for many of the newer OP insecticides. The O-isopropyl OPs are
less toxic to insects than their O-methyl and O-ethyl analogs. For example, parathion isopropyl is about
half as toxic to houseflies as either parathion-methyl or parathion-ethyl. However, the latter are
extremely toxic to honey bees, while the isopropyl analog is essentially nontoxic to these highly
beneficial insects. Unfortunately parathion iso-propyl also costs twice as much to produce as its analogs,
so it has never been used commercially.
Figure 5: Structure of malathion (left), its primary mammalian metabolite (center) and its activated
insect metabolite (right)
Biotransformation of Carbamates
Carbaryl is a fairly typical insecticidal carbamate, with an N-methyl and N-H group, and a bulky
(naphthyl) leaving group8. Esterases, including AChE, cleave the leaving group, resulting in a
carbamylated enzyme. In contrast to the phosphorylated ChE, the carbamylated enzyme is readily
regenerated, with destruction of a carbamate molecule.
Figure 6: Structures of carbamate pesticides
7
In fact, the only occupational poisonings with malathion occurred when malathion intended for use in a
malaria control program was improperly stored at high temperatures, resulting in generation of very
toxic impurities. Malathion itself has an LD50 > 1000 mg/kg in most mammals.
8
Carbamates in which the bulky group is the N-substituent and CH3 the leaving group often have
herbicidal activity, but are useless as insecticides.
A second degradative reaction is oxidative N-demethylation, which also results in irreversible carbamate
inactivation, without inhibition of AChE. Formaldehyde is produced as a byproduct of this reaction.
Quantities of formaldehyde resulting from carbamate intoxication are usually quite small, and as a
result its toxic effects are considerably less serious than those of AChE inhibition 9.
Carbamates can also be degraded via an epoxide intermediate, mediated by CYP 1A1. some carbamates
can also undergo S S=O reactions.
Other Reactions of Cytochromes P450
Aryl compounds – especially polynuclear aromatic hydrocarbons (PAHs) can also be hydrolyzed by the
P450s. The epoxide intermediate subsequently forms a trans-dihydro-diol which goes to a phenol. Such
multi-ring structure, if flat, can also intercalate into the DNA helix and (given the right structure) form
adducts with bases in DNA, especially guanidine.
The presence of a chemical that is metabolized by a given P450 usually leads to increased levels of that
isozyme, a process called enzyme induction. Enzyme induction increases the capacity of the organism to
metabolize endogenous and exogenous chemicals. It is a form of genetic derepression, causing increased
gene expression.
Such increased levels of oxidative enzymes result in increased levels of reactive oxygen species in the
organism: especially, but not only, in the liver. Reactive O may oxidize phospholipids and/or damage
membranes; it is thought that oxidation of membrane phospholipids is a cellular aging process. In
addition, it is possible that chemicals other than the xenobiotic causing the induction will be metabolized
by the increased isozyme, perhaps by pathways leading to different, and more dangerous intermediates
than the normal pathway produces. Many chemicals that cause cancer induce P450s; the correlation is so
pronounced that induction of P450 can be taken as a warning to examine the carcinogenicity of the
compound.
Figure 7: Benzpyrene (left) and its carcinogenic metabolite
Interspecies Differences
There are differences in biotransformation enzymes between species – both among higher and lower
vertebrates, and between classes. For example, evolutionarily similar P450s have different substrate
specificities in different classes of vertebrates, or even between rats and mice. CYP 1A1 is inducible in
fish, while CYP 2B1 is not; both are inducible in rats and humans.
9
However, if methanol is ingested in quantity, formaldehyde levels in the optic nerve can build up to
destructive levels, causing destruction of the nerve and permanent blindness.
In many cases males and females of the same species differ in levels (or even presence) of P450
isozymes. Examples: aromatase, which catalyzes conversion of testosterone to estradiol. In the rat, it is
present in the male testes and the female adrenal glands. As sperm matures, it Apparently requires pulses
of estradiol, provided by pockets of aromatase in the testicular cells. Another example is CYP 3A1,
which is present in 25 day old rats of both sexes, but disappears in the adult female. In mice, females
appear to maintain CYP 3A1. Finally, mammalian embryos – which can depend on the maternal
organism to process wastes – do not produce active P450s until late in prenatal development. In contrast,
chick embryos, which develop independently within a hard shell that allows for neither uptake of
nutrients nor excretion of solid or liquid wastes, have active P450s by the end of organogenesis.
Enzymes other than P450s also differ between species. In insects there are no carboxyesterases capable
of cleaving the alkyl moieties of malathion; in mammals, there are. This difference in metabolic capacity
makes malathion one of the safest insecticides in commercial use. Such differences have been exploited
not only in pesticide design, but also in drug design. Moreover, in many cases it is possible to add
metabolic blocking agents to retard the degradation of a drug or pesticide.
Both nutritional status and the presence of contaminants may greatly affect the toxicity of a pesticide,
often in unexpected ways – for example, the presence of small quantities of isomalathion vastly
increases the toxicity of malathion to mammals.
Most P450s are membrane-bound to the endoplasmic reticulum (ER). This means that fat-soluble
chemicals have access to these enzymes, while water-soluble compounds probably do not.
Methylenedioxyphenyl compounds have been shown to potentiate pesticides that are metabolized by
P450s. The primary congener used to potentiate insecticides is piperonyl butoxide, which binds to P450
and inactivates it. Moreover, when oxidized by P450, piperonyl butoxide releases carbon monoxide,
which is itself an inhibitor of P450s. note, however, that prolonged exposure to piperonyl butoxide
results in induction of P450, probably in a feedback loop response to the initial inhibition.
Figure 8: Structure of the insecticide synergist piperonyl butoxide, which prolongs the action of
many insecticides by inhibiting cytochrome P450.
Piperonyl butoxide has been used to synergize both OPs and carbamates, but has proved most valuable
in prolonging the action of pyrethrum, a derivative of Chrysanthemum cineraraefolium. Pyrethrum has a
very rapid knock-down action which is invaluable (for example, in formulating anti-wasp insecticides).
Unfortunately its action is so brief – it is metabolized so rapidly – that the initial ‘knockdown’ is often
not lethal. Combining pyrethrum with piperonyl butoxide prolongs the neurotoxic action of pyrethrum
long enough to kill the target.
Other chemicals that inhibit P450s include sesame oil and its derivative, sesamex.