Vaccari D.A., Strom P.F., Alleman J.E. Environmental Biology for Engineers and Scientists

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18.5.1 Phase I Reactions

Phase I reactions include oxidation, reduction, and hydrolysis. These reactions may pre-

pare xenobiotics for the phase II reactions.

Oxidation Toxins can be oxidized by numerous pathways. However, the most important

system is the group of enzymes called monooxygenases, containing the cytochrome P450

enzyme system (also called mixed-function monooxygenases). These are nonspeciﬁc

membrane-bound enzymes found mainly in the endoplasmic reticulum of the liver, but

also in other tissues. Their function is to take one of the oxygen atoms from an O

mole-

cule and insert it into the toxin molecule while the other is combined with two protons to

produce H

RH þ O

þ NADPH

) ROH þ H

O þ NADP

Speciﬁc examples are shown in Figure 18.3. Although not shown in the ﬁgure, all of

these reactions involve NADPH

and O

as reactants and monooxygenase as a catalyst.

O R

ROH

phenol

dealkylation

epoxide

epoxidation

C C R

epoxide

deamination

C CH

hydroxylation

Figure 18.3 Microsomal oxidation biotransformation reactions.

748

FATE AND TRANSP ORT OF TOXINS

One important effect of cytochrome P450 monooxygenases is to convert aromatics

into epoxides. It is thought that this metabolite is responsible for the cancer-causing beha-

vior of aromatics such as benzene and benzo[a]pyrene. The epoxides are unstable and

convert readily to phenols. Phenol is found in the urine of persons exposed to benze ne.

Other enzymes can convert epoxides into dihydrodiols, which are nontoxic and easily

excreted.

Some chemicals, called inducers, increase the rate of biotransformation of other com-

pounds by stimulating the synthesis of more cytochrome P450. Thus, these substances are

likely to interact with other substances in a more than additive manner (Section 19.5).

Table 18.2 lists some of these compounds.

Other compounds inhibit cytochrome P450 and thus may interact in a less than additive

manner with other substances. Some of these are listed in Table 18.3. The effect of indu-

cers and inhibitors can be used to show whether a substance or its metabolite is causing

the toxic effect. For example, bromobenzene causes necrosis in the liver, which is

increased by phenobarbital and decreased by other inhibitors. Therefore, the damage

must be caused mostly by a metabolite.

The liver enzyme alcohol dehydrogenase converts ethanol into acetaldehyde, which is

even more toxic than ethanol. This is followed b y the action of aldehyde dehydrogenase,

which forms easily excreted acids.

Reduction Under low-oxygen tension conditions, the cytochrome P450 monooxy-

genase system is capable of catalyzing reduction of azo and nitro compounds to amines

(Figure 18.4). Other types of compounds that are reacted reductively are those containing

an aldehyde, ketone, disulﬁde, sulfoxide, quinone, N-oxide, or alkene group. Aldehydes

are converted to alcohols and ketones to secondary alcohols.

Another reduction mechanism is the replacement of a halogen bonded to a carbon by

a hydrogen (reductive dehalogenation). This is responsible for the hepatotoxicity of

carbon tetrachloride and similar halogenated alkanes. (Dehalogenation can also be

TABLE 18.2 Pollutants and Other Substances That Induce Cytochrome P450

Drugs Industrial Chemicals PAHs

Barbiturates Aldrin/dieldrin Lindane Benzo[a]pyrene

Ethanol Chlordane PCBs Dibenzanthracene

Nicotine Chloroform Piperonyl butoxide 3-Methylcholanthrene

Steroids DDT, DDD Pyrethrum

Heptachlor Urethane Ketones

Toxaphene

Source: Williams and Burson (1985).

TABLE 18.3 Substances That Inhibit Cytochrome P450

Piperonyl butoxide Aminotriazole Carbon tetrachloride

Chloramphenical a-Napthyl isocyanate Bromobenzene

Cobaltous chloride Carbon disulﬁde

Source: Williams and Burson (1985).

BIOTRANSFORMATION

749

performed oxidatively by replacing a hydrogen and a halogen with oxygen, or by

eliminating two adjacent halogens and forming a carbon–carbon double bond.)

Hydrolysis Esters, epoxides, and amides are subject to hydrolysis catalyzed by a variety

of enzymes (Figure 18.5). One important group is the cholinesterases, which include

acetylcholinesterase. This was described above, although in the context of the toxic effect

of pesticides. Carboxylesterases bind or hydrolyze organophosphorus pesticides. Epoxide

hydrolase converts epoxides, such as those formed by cytochrome P450 enzymes, into

diols. This protects against the genotoxic effect of the epoxides.

18.5.2 Phase II Reactions

Conjugations These phase II reactions involve combining the toxin with another mole-

cule or functional group. These usually increase polarity, and therefore solubility, to faci-

litate excretion. Phase II reactions usually take place in the cytoplasm rather than in

microsomes. They tend to be faster than phase I reactions, making the latter a rate-limiting

step.

The most important detoxiﬁcation mechanism is the conjugation with glucuronic acid

(Figure 18.6). The enzyme responsible for it is found in the endoplasmic reticulum of

many tissues, but especially in the liver. Many classes of compounds can be glucuroni-

dated, including aliphatic and aromatic alcohols and amines.

Other conjugations involve addition of amino acids such as glutamine or glycine, or

sulfate, methyl, or acetyl groups. The enzymes for these processes are found in the

endoplasmic reticulum and the mitochondria, but mostly in the cytoplasm of hepatic

cells. Methylation differs from the other phase II conjugations in that it tends to reduce

aqueous solubility rather than increase it. The methyl group is usually added to a heteroa-

tom (O, N, or S). A rare exception is the methylation of a carbon in benzo[apyrene to form

6-methylbenzo[a]pyrene.

RCH

CCH

RCH

CCH

RCH

Figure 18.4 Reduction biotransformation reactions.

750

FATE AND TRANSP ORT OF TOXINS

One of the most important detoxiﬁcation mechanisms is the formation of mercap-

turic acid derivatives. This mechanism acts on a wide variety of xenobiotics. The

reaction sequence starts with conjugation of the toxin with glutathione. Glutathione is a

tripeptide:

CH CH

NCH

C N CH C N CH

COOH

OOH

glutathione

-glutamate

glycine

cysteine-SH

a) Aliphatic ester

COR

esterase

RCOH

ester

acid

alcohol

b) Organophosphate ester

POR

esterase

POH

organophosphate ester

acid alcohol

c) Peptide bond

CNR

amidase

COH

acid

amine

amide or peptide

epoxide

hydrolase

OH OH

epoxide

hydrolase

HOH

d) Epoxide

Figure 18.5 Hydrolysis biotransformation reactions.

BIOTRANSFORMATION 751

Notice the sulfhydryl (SH) group on the cysteine. It is this that is bonded to the

foreign molecule in the ﬁrst step. For example, consider the reaction of glutathio ne

with 1-chloro-2,4-dinitrobenzene shown in Figure 18.7a. The second step is remov al of

the two free amino acids leaving a thiol ester with cysteine. This is followed by the

third and ﬁnal step in Figure 18.7b, a reaction with acetyl-CoA to produce 2,4-dinitrophe-

nyl mercapturic acid. This mechanism is effective with electrophilic compounds . It is an

important detoxiﬁcation mechanism for elimination of carcinogenic epoxides produced

by the monooxygenases.

Glucuronic acid conjugation

Sulfate conjugation

Methylation

OCH

Amino acid conjugation

COH

NCH

COH

glycine

benzoic acid

+ H

COOH

+ H

phenol glucuronic acid phenylglucuronide

Figure 18.6 Conjugation (phase II) biotransformation reactions.

752

FATE AND TRANSP ORT OF TOXINS

Bioactivation may involve a complicated series of reactions in several tissues. For

example, 2,6-dinitrotoluene is ﬁrst oxidized by cytochrome P450 and conjugated with

glucuronic acid in the liver. It is then excreted in the bile, where bacteria reduce one or

both nitro groups to amines and split off the glucuronide. These are reabsorbed by the

intestines and return to the liver. The amine group is then hydroxylated by P450 enzymes.

The resulting compound can react with DNA, causing mutations that can lead to liver

cancer. Table 18.4 lists some compounds that are bioactivated.

18.6 EXCRETION

Compounds that are not biotransformed or stored are excreted to the exterior of the organ-

ism. It is remarkable that the body can do this for many xenobiotic compounds as well as

natural ones. The kidney is the most important organ for excretion of toxins or their

metabolites. The liver is next in importance, followed by the lungs. Lesser roles are

played by the hair, skin, sweat, nails, and milk.

As you may recall from Chapter 9, the kidney works by ﬁrst passively ﬁltering about

125 mL/min of plasma from the 6 00 mL/min of blood perfusing it. This passes solutes and

macromolecules up to molar mass 60,000, thus retaining cells and most plasma proteins.

Then, by tubular reabsorption involving both active and passive transport, the nephr on

conserves water, several sal ts, and other nutrients, such as sugars and amino acids, and

forms concentrated urine. Finally, tubular secretion actively transports wastes, including

salts, drugs, and toxins. The ﬁnal urine ﬂow is about 1.0 mL/min.

SCH

CCCoA

COOH

NH C CH

+ CoA

acetyl-CoA

2.4-dinitrophenyl mercapturic acid

COOH

+ γ-Glu-CySH-Gly

SCys

γ-Glu

Gly

+ HCl

2,4-dinitrochlorobenzene

glutathione

(a)

(b)

Figure 18.7 Glutathione conjugation (phase II) biotransformation reaction.

EXCRETION 753

The rate of excretion (mass per unit time) divided by the concentration in the blood

is called the renal clearance. If a toxin is ﬁltered, but neither reabsorbed nor secreted,

the full 125 mL/min are ‘‘cleared’’ of toxin. If the toxin is partially absorbed and not

secreted, the renal clearance will be less than 125 mL/min. Toxins that are actively

secreted can have a renal clearance as high as the perfusion rate of 600 mL/min.

Lipophilic toxins tend to be protein bound in the blood, and thus are retained at the

ﬁltration step. Those that are dissolved in the plasma are fairly easily reabsorbed. Active

transport can excrete even those compounds that are bound. For example, it was found

that DDA, a polar metabolite of DDT, was excreted 250 times as rapidly as DDT in

mosquito ﬁsh, although both were protein bound to a similar extent. The fact that DDA

was being actively transported was established by the observation that metabolic inhibi-

tors decreased its excretion rate. The DDT, on the other hand, was probably being reab-

sorbed by passive diffusion.

Ionic molecules have less tendency to be reabsorbed. Therefore, excretion of weak

acids and bases will depend on their dissociation constant and the pH of the urine.

Urine is usually acidic, making passive diffusion more important for organic bases than

for organic acids. Excretion of acids has been manipulated therapeutically by increasing

the pH of the urine by ingestion of bicarbonate. This increases the ionization of the acids,

preventing their reabsorption. In addition, the nephrons have separate mechanisms for

active secretion of organic acids and bases. Biotransformation by the liver and other

TABLE 18.4 Bioactivated Compounds

Parent Compound Toxic Metabolite Toxic Effect

Allyl formate Acrolein Hepatic necrosis

Benzene Benzene epoxide Bone marrow depression

Bromobenzene Bromobenzene epoxide Hepatic, renal, bronchiolar

necrosis

Carbon tetrachloride Trichloromethane free Hepatic necrosis, hepatic

radical cancer

Carcinogenic alkylnitrosamines a-Hydroxylation Hepatic cancer

Carcinogenic aminoazo dyes N-Hydroxy derivatives Hepatic cancer

Chloroform Phosgene Hepatic, renal necrosis

Ethanol Acetaldehyde Varied

Fluoroacetate Fluorocitrate General toxicity

Halothane Free radical Hepatic necrosis

Hemolysis-producing aromatic N-Hydroxy metabolites Hemolysis

amines

Methanol Formaldehyde Retinal and general toxicity

Methemoglobin-producing aromatic N-Hydroxy metabolites Methemoglobinemia

amines and nitro compounds

Naphthylamine N-Hydroxy naphthlamine Bladder cancer

Nitrates Nitrites Methemoglobinemia

Nitrites plus secondary or tertiary Nitrosamines Hepatic, pulmonary cancers

amines

Oleﬁns Epoxides Skin cancer

Parathion Paraoxon Neuromuscular paralysis

Urethane N-Hydroxyurethane Cancers, cytotoxicity

Source: Lu (1991).

754 FATE AND T RANSPORT OF TOXINS

organs serves to increase excretion by the kidney by increasing the polarity of the toxins.

They also tend to lower the pK

of o rganic acids, increasing their ionization.

Besides its role in biotransformation, the liver excretes directly via bile. The liver

tends to be more effective than the kidney in excretion of protein-bound compounds

and compounds with molar mass greater than about 300 to 400. The liver has separate

active transport mechanisms for organic acids, bases, and neutrals, and possibly a separate

system for metals.

Although most toxins excreted in the bile are eliminated with the feces, many can be

reabsorbed in the intestines. All the blood from the intestines passes through the liver

before going elsewhere in the body; thus, compounds can be reexcreted. This results in

a cycle called the enterohepatic circulation. This is especially true of lipophilic com-

pounds. In such cases, elimination can be facilitated by ingesting an adsorbent such as

activated carbon. This will compete with the intestines to absorb the toxin and then

carry it out with the feces. Sometimes, lipophilic compounds which have b een rendered

more soluble by conjugation reactions in the liver can have the bonds hydrolyzed by

intestinal microorganisms, increasing their reabsorption.

The importance of excretion by the bile has been demonstrated by the observation that

toxicity can increase severalfold in animals with experimentally blocked bile ducts. For

example, this increases the toxicity of diethylstilbestrol (DES) to rats by 130 times.

The lungs are important in excretion by the passive diffusion of compounds with high

vapor pressure or Henry’s constants. This means smaller, relatively nonpolar molecules

such as hydrocarbons and chlorinated hydrocarbons. Soluble compounds such as ethanol

are found if the blood concentration is high enough. Thus, the concentration of chloro-

form in exhaled breath is proportional to the blood concentration. Since storage in

depot fats acts as a reservoir for these compounds, they may be detected in the breath

long after exposure has ceased. In ﬁsh, excretion through the gills also seems to be

most important for lipophilic compounds that are not biotransformed rapidly.

Metals are concentrated on hair. Although in humans this is not an important excretion

mechanism, it provides an easy way to test for exposure. In fact, since hair grows about

1 cm per month, it is possible to test segments of hair to obtain an indication of the time

history of exposure. Excretion by hair is important in furred animals, and this must be

taken into account when attempting to extrapolate to humans from laboratory tests on

rats or other small mammals.

Due to its fat content of 3 to 5%, milk can be important for excretion of lipophilic

compounds. These include chlorinated hydrocarbons such as DDT and PCBs. The pro-

cesses that transport calcium into the milk are thought to do the same for lead. Unfortu-

nately, this route of excretion is more important as a source of these contaminants for

nursing young than as a way of clearing the toxicant from the mother.

18.7 PHARMAKOKINETIC MO DELS

A model is a kind of analogy. It is a surrogate for a real proce ss that is easier to analyze

and experiment with. A mathematical model is thus a set of equations that behave

similar to a real system. Often, the process of developing a mathematical model helps

to develop our mental model, leading to improved understanding. Furthermore, the

model may be useful in making predictions, and for designing engineered systems to

obtain desired results.

PHARMAKOKINETIC MODELS 755

Ultimately, we would like to predict actual toxic effects. The models described here,

however, will only predict the exposure of an organism to substances in the environment

and the concentration of various tissues that results. Our interest will ﬁrst be in pharma-

cokinetic models, models of the behavior of toxic substances within organisms: how they

are taken up, distributed and stored, biotransformed, and excreted. The same principles

can then be used for environmental models, models of the fate and transport of toxic

substances between and among compartments of the environment (air, water, soil, etc.,

and different organisms in the environment), which control exposure.

A common type of model is the compartment model, which represents organisms by

one or more volumes characterized by a single concentration for each substance being

modeled and which is physicochemically and biologically uniform. For example, an

organism could be represented by three compartments: the gut, systemic ﬂuids (blood),

and target organs. The compartments are connected to each other and to the environment

by transport mechanisms. Biochemical reactions create or consume the modeled sub-

stance within the compartments. Mathematical relations describe the rate at which each

of these processes adds or removes material from each compartment. The process of

model development can be broken down into the following steps:

1. Deﬁne the compartments that will make up the model.

2. Identify the transport mechanisms that move substances between compartments.

3. Identify the biochemical reactions producing or destroying the modeled substance

in each compartment.

4. Write the mathematical description of the individual transport ﬂuxes and reaction

processes.

5. Link the processes with material balance considerations to produce the ﬁnal

mathematical form of the model.

6. Mathematically solve the mode l using the appropriate initial and boundary condi-

tions.

7. Validate the model by comparing with experimental results.

Material balance expresses the law of conservation of matter. The balance of the

matter that enters or leaves a compartment, or is created or destroyed within it, must result

in an accumulation. This can be expressed in this way:

accumulation ¼  inputs þ  outputs þ  reactions ð18:13Þ

The accumulation term is the change in mass, M, within the compartment, which in turn is

the product of volume, V, and concentration, C. All the terms in equation (18.13) should

have the units [M t

1

]. It is common to assume that the volume of the compartment is

constant (no growth). In this case, the accumulation can be expressed in terms of the

change in concentration in the compartment:

accumulation ¼

dðVCÞ

¼ V

ð18:14Þ

The ﬂuxes are the mass transport ﬂows moving matter from one compartment to

another, or to or from the environment. These include the various forms of absorption,

756 FATE AND T RANSPORT OF TOXINS

such as passive diffusion [equation (18.11) or ﬁlm theory equation (18.12)]. An important

type of ﬂux is advection, the result of a substance being carried into a compartment by

ﬂuid ﬂow. Examples include transport of a toxin to the liver by blood ﬂow, or water ﬂow-

ing through the gills of a ﬁsh. Advective ﬂux, F, can be represented by the product of ﬂow,

Q, and concentration:

F ¼ QC ð18:15Þ

Advective ﬂux can also describe uptake by ingestion, wherein the ﬂow is the mass of

food ingested per unit time, and the concentration is the average toxin concentration in the

food. Fluxes must have positive signs for transport into a compartment, and negative for

movement out.

The biochemical reactions are described using the methods from chemical kinetics.

The general form of the reaction term is

R ¼ Vr ð18:16Þ

where r is the reaction rate , that is, the mass produced or consumed per unit volume per

unit time. The rate is described, in turn, by a rate law, which gives the dependence of the

rate on other factors, especially concentrations of reactants. Often, simpl e elementary rate

laws can be assumed. In some cases more complex kinetics may be assumed, such as a

rate based on Michaelis–Menten kinetics [equation (5.36)], or one of the other rate equa-

tions from Section 5.3.1. Here we describe two of the simpler kinetic rate laws, which are

often sufﬁciently accurate to describe many reactions. The ﬁrst is the zero-order reac-

tion, in which the rate is constant independent of the concentration of reactants:

r ¼k ð18:17Þ

The sign will be positive for production reactions and negative for destruction. An

example of a zero-order reaction is the metabolism of ethanol in humans (Problem 18.1).

The other simple elementary rate law is the ﬁrst-order reaction, in which the rate of

reaction is proportional to the amount of reactant present:

r ¼kC ð18:18Þ

First-order reaction is probably the most com mon rate law. The negative form is called

ﬁrst-order decay. Its use will lead us to several important parameters for describing

toxicant behavior which are developed in the next two sections.

Models developed as just describ ed are differential equations which, when solved with

appropriate initial conditions, predict the concentration of toxin in a compartment vs.

time. Simpler models can be solved analytically giving the result as a function. More

complex models must often be solved numerically using a computer, giving the concen-

tration vs. time as a table of values. Models that predict how results change with time are

called dynamic models.

Often, if enough time is allowed to elapse, dynamic models show that concentrations

approach a constant value. In the limit as time goes to inﬁnity, all changes cease, and a

steady-state condition exists. Mathematically, the steady-state condition could be found

by taking the limit of an analytical solution as time goes to inﬁnity. Even simpler, and

PHARMAKOKINETIC MODELS 757