Bioactivation or inactivation of toxic compounds?

TIPS - March, 1980

I ‘76 .

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phase 2 (conjugation) reactions. Umil recently. it was generally believed that only phase I reactions could generate reactive metabolites, whereas products formed during phase 2 reactions were harmless. This has now been shown in many cases not to be true. The quantitatively major part of phase I metabolism is carried out by the microsomal mono-oxygenase system’ (see Fig. I). A cytochrome P-450 enzyme, being the terminal component, accepts two electrons from either NADH or NADPH and introduces one oxygen atom into the substrate. Recently, it has been possible to reconstitute the system in vesicles from purified phospholipid components and to establish the participation of cytochrome bq2. The cytochromes P-450 are hemoproteins which preferentially accept hydrophobic substrates and carry out (I) epoxidation of double bonds and aromatic ring systems, (2) hydrbxylation of C-H bonds in the preferential sequence tertiary> secondary> primary and (3) hydroxylation or oxidation of heteroatoms bearing a proton’. They have molecular weights of about 50,000 and they are integral membrane proteins. However they contain only about 45% hydrophobic amino acid residues. Evidence is accumulating to support the idea that the major part of the enzyme molecules, including the active sites, is localized outside the membrane. The N-terminal domains of the P-450 proteihs so far characterized consist of amino acids, probably hydrophobic forming the membrane-binding anchor of the proteins. Lipophilic compounds have to diffuse from the bilayer to the hydrophobic heme pocket localized outside the membrane before subsequent metabolism is possible. The phase 2 reactions are attributed to the action of a series of microsomal and

’

_.

Two of a ltind is concerned on this occasions with sot?te interesiing uspecls qf the tosico/og_vof drugs and orher xenobiorics. Ingehltan-Sundberg shows how .WW subsiancus produce adverse effects on1.vafter the-v have been chetnitul!,~ tnod(fied in Ute body and bleidenber): describes how adverse effects qf drugs somerimes nkric rhe s.~tptoms of idiopa!hic

ldiseases.

ioactivation or inactivation of toxic compounds? Magnus Ingelman-Sundberg We are conrinuously exposed to a huge amouru of foreign chemicals, many* exerting acGvaGon 10 reacfive intermediares. The net !oxic effect will be ajknction of rhe relative activiries of roxificarion and detoxification reactions. This balance will be affected rE>yseveral factors such as exposure to environmenlal contrrminants. nutrition, generic status of rhe individual, supply of cofactors and the do:‘e of the toxic compound in question. Ioxic effects in rhe b 3d.v after rnerabolic

0ur environment IS contaminaied by more than four milhon different synthetic compounds. According to estimations performed by the E;nvironmental Protection Agency and ihe Food and Drug .4dministration more than 63,000 chemicals are in commozl use. These include :,arious drug constitlIsnts, food additives, pesticides and hair d) es. Those chemicals which are not water soluble have to be moditied in our body to more polar compounds. before eificient excretion is possible. This cheptical modification is carried out by the drug-metabolizing enzymes, preferentially located in the i:,ver. but also in the lungs, the kidneys, the skin and the gilstro intestinal tract. During the biotransformation chemically.

‘very reactive intermediates are often formed which either uncouple integrated biochemical processes or combine covalently with various macromolecules such as DNA, RNA and protein. It is now evident that serious toxicities, such as carcinogenesis, mutagenesis and cellular necrosis as well as methemoglobinemia, hemol>?ic anemia, blood dyscrasias and hypersensitive reactions are often attributable to the action of such reactive intermediates. The microsomel drug-metabolizing system

The biotransformation of environmental chemicals involves so called phase I (oxidation, reduction, hydrolysis) and

/ epoxida hydratasa

0, I ,I NAOPH-NADPH-cytcxhrowa

P-450 reductase

Eieccron transport componenrs

,mchroma

P 450

SUBSTRATE

\ ‘\_.

Hz0

.’

/,

R@;,~E

gllIcurOnyltraw3feraaa

Fit. 1. ThedruR-merubokingsystem.

/

.\\W//,,/ INTERMEOI~ //I

I I W’., I

-tra

\

glutathiona

transferam

-1

TIPS

- March.

wluhle

I980

I--

conjugating

enzymes’ (see Fig. I).

Most environmental tially

eliminated

combined

toxicity

chemicals are essen-

from

the body

t

by the

action of phase 1 and phase 2

metabolizing

enzymes.

Exceptions

substances of a highly non-polar

are

= 0-HCOOH

nature,

such as polychlorinated biphenyls and hexachlorobenzene, which are biologically

stable

tendency

in

the

body

due

to be deposited

adipose

tissues.

spectrum

The

to

their

unchanged

extremely

of compounds

LH

L &

lipid peroxidation

in

broad

acting

1

as sub-

toxicity

strates for these enzymes is possible due to the presence of multiple type of enzyme.

forms

forms of cytochromes

P-450, seven forms

of glutathione-S-transferases, of

of each

At least seven different

sulfotransferases

and

glucuronyltransfetases

five forms two

forms

of

have been found

diet

more

efficientI>

metabolize

some

drugs than people having con\entionall>

cooked food’. The rate and nature of the biotransformation

of different

chemicals

and partially characterized. The enzymes exhibit different but partially overlapping

will hence be dependent on the type of environment to which the individual is

specificities

exposed. This is probably

and can thereby accept sub-

strates having

a diversity

Teleologically,

it is easy to understand

that nature

of structures.

has solved this problem

by

for

the

differences

in drug

logical

completely

importance.

enzyme.

form

of

the

interindikidual

metahohzinp

capa-

city among human\ and experimental animals. In addition, genetic and ph>\ie

having multiple forms of each kind of enzyme instead of one single and non-specific

one explanation

pronounced

factors

are

of

iour5e

cf

The latter type of catalyst must

necessarily

exhibit

low

affinity,

wotild be a severe handicap small concentrations

which

in removing

of toxic chemicals

Reactive intermediates The reactive intermediates produced bl; metabolic activation of various toui<

from the body. This can be overcome by

compounds are of two kinds: electrophils

multiple forms with higher specificity and

and radicals” (see Table I). Example5 of the former compounds are epolidcs.

therefore higher affinity Suggestions

for the substrate.

have been made

that

cyto-

carbcnium

ions and nitrenium

ions. Onl!

chromes P-450 are similar in nature to the

a few radicals

immunoglobulins,

posed as reactive intermediates.

i.e.

genetic

informa-

tion should exist making possible the bio-

form

synthesis of huge amounts

undergo

P-450s

exhibiting

specificities.

substrate

dependent

the >,pecificities of

respective

vario:ls

However,

the cytochromes

P-450 overlap to such an

extent that 10-15 different most probably cover environmental

of different

rypes of P-450 hydrophobic

of a given compound

from the body will be dependent

on the

levels of the various enzymes in the tissues and the availability Many

xenobiotics

inducing

one

metabolizing barbital induction

of suitable cofactors. have th.r capability

or

more

another

of

of

drug-

enzymes.

Thus, e.g. phenoto rats results in

of certalin types of cytochrome enzyme

enzymes, whereas

Ipattern is produced

e.g.

3-methylcholanthrene

tion.

Polyaromat’ic

a lot of

the

administration

P-450 and conjugating

hydrocarbons

drug-metabolizing

by

administrainduce

enzymes

in

man. Thus, smokers will eliminate certain drugs addition,

faster

than

CHCI,

a reductive

cytochrome

homolytic

are

not

to

non-smokers.

In

people having a charcoal broiled

CI,C-

toxic

induce

subsequent in

P-458

itself,

per lipid

2).

F,C-

se. but

since

are

peroxidation of cellular

This damage

destruction

membrane, integrated,

The

and

destruction

(Fig.

ma! P-GO-

cleat age.

products,

results

fatty

Chloro-

also halothane

thought with

been pro-

perhaps

components

chemicals.

The elimination

and

have hitherto

albo

cf

cytochrome

the

microsomal

in which cytochrome P-49 IZ contains many tmsaIurated

acids

Nhlsh

upon

prroltdatlon.

efficiently destroy the P-450 heme‘. Not much is known about Hhiih rlecrosli. cellular mechanisms cause interreactive electrophilic Clearly, mediates react cotalently sith nu~lc.~,philic sites on DNA and RNA. 11 hen studying acetaminopheninduced

hepatic

and furosrmide-

necrosis

it

has

possible, by means of autoradiograms. correlate reactive

the

covalent

metabolites

liver preferentially

binding

oi

bec’n IO

t;lc

to the areas in t?e

being destroyed.

TIPS - Matvh, IWO

178 TABLE 1. !?easri. r’intermediates :n the mrlrbohslm of foreign __.

_-

Cornpow

COJ’IWUnds

--

_--

-..-

Intermediate formed by the action of

Proposed reactive intermediate

Formula

Type Iof toxicity

0

Q

; CH,

cytochrome P-W

hepats,;necrosis rcnnllnecrosis

cytochromc P450 and epoxidc hydratase

carcip.3gcnesis

cytochrome Pd50

hcpatic necrosis lung necrosis

cytochrome P-450

hepatic necrosis

cytochromc P-450

hepatic necrosis renal necrosis

cytochrome P450

blood dyscrasias

cytochromc P-430

hepat ic necrosis renal necrosis

cyrochrome P-450

carcinogenesis

cytochromc P-450

hepatic necrosis renal necrosis

Halothane

cytochrome P4SO

hepatic necrosis

brkzid

rransacetylase and cytochrome P450

hepatic necrosis

cytochrome P-450

hepatic necrosis

cytochromc P-450

hepatic necrosis

cytochrome P-GO and sulfotransferase or glucuronyltransferase

hepatotoxicity

cytochrome P-450

hcpatic necrosis neoplasia

cytochrome PdSO and alcohol dehydrogenase

lung toxicity

ox

Acetamincphm

-

=N

OH

Br

0‘1

Bromobenzene

0

*,

s=c=s

Carbon disulfide

s: b.

Cl Carbon tetrarhlondc

Cl

c-

CL

c-0

OH Chloramplwnicol

Cl

Cl

Cl CHtOH

- CH - &

0

OH

H

- NH - 6 - 6 -Cl

O,N-

/ ’ Q

Cft,OH

-6”~dH-NH-&&C,

0

0

2, 0

H CI2

Cl-C-Cl

chloroforrrl

iI

Cl CHJ,

Dimethylnwosoamim

CH;

N-N=0 CH,’

Furosemidc

- CH,-

NH-

’ ’

Q

-SOINH,

0

‘F H

CktJ

H-&3

lprostiazid

b-i,

Parathson

tC,HJOtJ-

b- 0 -

.s:.

/ \ INO,

0-

0 Phmaca,r

Thit2aceti~midc

xykne

o=

+H,-O-ION”-:.-C”, -

Ctf, - C - NH,

H,C-

0-’ /

-CH,

Q -

=N-&CH,

CH, -i-

NH2

TIPS - March, I980

i-9 suifo

philic enough, e.g. carbon atoms bearing an epoxide are

group

conjugated

or

a halogen

with

glutathione.

specificity of the glutathione a

hydrophobic

spontaneous, An the

factor

soluble

reaching

sulfotransferases

nuclear

them.

The and

diffusion

the

with

therefore

formed

in the membrane

by

induction

the

the halobenzene

lateral

without

being

in contact with the cytosolic conjugating enzymes.

The

tetrachloride

toxic

action

of

carbon

is thought to be potentiated

by its lipid

solubility

%methylcholanthrene

the

by P-450

nucleus

in the 3,4-epoxidation

of bromo-

benzene are induced by this drug, whereas

endoplasmic

contact

intermediates reach

pating

hydrophobic may have difficulty

envelope

could

are

i.e.

is in direct

reactive

is

conjugating

most of the glutathione

and

enzymes,

reticulum

determining

the

reactive intermediates in

CM

conjugation

in

of

Firstly,

transferases,

-NW6

0

even

of a reactive intermediate

availability

reagent.

the

1’

P-4w

takes place.

important

detoxification

on

In addition,

non-enzymatic

with glutathione

El-O-

carbon atom

domain

substrate are required.

0

transferases

is low; only an electrophilic and

The

/ ,--Et-o/‘-NO”.&CH,--Et 0~ /i-N 0 phiA” 0 0-I \0 so, transferase

atom,

because its acces-

of

treatment

P-450

enzymes

derivative

toxic 2.3-epoxide. in

humans

to the non-

Isoniazid

be more hepatotoxic

results in converting

is known

at therapeutic

with

the

fast

to

doses

acetylator

phenotype since these subjects will produce more acetylhydrazine. i.e. the precursor of the reactive intermediate above) than do slow acetylators

(cf.

. (2) The

which are the substrate for carbon tetra-

levels of the conjugating enzymes are known to be influenced by various drugs

chloride-induced

and may thus determine the detoxifcation

sibility

to

the

relatively

lipid

high.

reactions,

is

impaired

of certain

e.g.

by

conjugation

depletion

of

the

In the case of e.g. acetamino-

phen or bromobenzene

administration

animals, low doses are non-toxic high doses result thione

acids

peroxidation.

Secondly,

saturation

cofactors.

fatty

may adso originate from the

detoxification ready

unsaturated

in

the

in depletion liver

dramatic

increase

reactive

electrophils

comitant

induction

with

in

of glutasubsequent

the and

to

whereas

amount thus

a

of con-

of hepatic necrosis. It

appears that in the liver glucuronidation has

a

high

conjugations sulfation

calpacity, have

has low

glutathione

medium capacity

extent of acrtylation

capacity, whereas

the

is variable depending

upon the genetic status of the individual.

process. The toxicity

originating

from

the absence of the specific type of glucuronyltransferase

in the newborn.

(3) The

relation between the level of cofactors and reactive of

intermediates

great

detoxification. rather

will of course be

importance

in

Despite

in aerobic animals

cells (-IO-

wirh

determining the

high concentration

normalla

of glutathione treatment

’ \I)

Lysteine,

covalent the

will decrease both binding of acetaminophen

drug-induced

contrast,

hepatic

depletion

of

potentiates

and

binding.

between compotmds ment

of

sulphation

Competition

for conjugation of

In b!.

hepatotosGt>

(4)

phenomenon.

simultaneous

to the and

necrosis.

glutathione

diethylmaleate covalent

of

a precursor

glutathione,

well-known

What determines the toxicity?

of chlorarnphenlcol

is high in the neonate,

The

IS a

impair-

isoprenalinr

b)

administration

of saiisjla-

mide leads to a pronounced

pofenriation

steady-state level of a given reactive inter-

of its cardiovascular

In a slmllar

mediate and thus the toxicity of the parent

uay,

compound.

cylamide

A number of factors will influence the

(I) The level of the activating

enzymes. of

e.g.

In the

parti,cipating

other

words,

specilfic

the amount

form

in the metabolic

of

P-450

activation

concurrer

induce

different

or

P-450.

Thus,

hepatotoxic phenobarbital

cytochrome

brolmobenzene in

animals

is highly

treated

since PASO-forms

with partici-

greatly

formed. In conclusion, importance

of

t administration enhances

of sali-

acetamino-

by necrosis phen-induced hepatis decreasing the amount of phenalsulfate

is of fundamental importance. As mentioned above, various xenobiotics forms

effects.

very many factors are of

in determining

inactivation

of

detosification

a toxic

compound.

There still appears Io be no better \ray of preventing toxic effects than to keep away from all dangerous chemicals.

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