Environmental Health and Toxicology

Chapter 3 Metabolism of Xenobiotics (foreign compounds)

外来物 的 新陈代谢

Phases of MetabolismOnce xenobiotics enter our body, most will be excreted 排泄 or metabolic inactivation 灭活 as quick as possible.

Water-soluble xenobiotics will be excreted through the kidneys 肾 as urine 尿， no metabolism required.

Lipophilic 亲脂性 xenobiotics will be excreted by biliary excretion 胆汁中的排泄 or undergo metabolism to become more polar 極性 and then water-soluble and excreted by the kidneys.

Two phases in the metabolism of xenobiotics

1.Biotransformation 生物转化 which involves oxidative 氧化 reactions

2.Conjugation reaction 结合反应 which involves combination with highly water-soluble moieties一部分或一半

Biotransformation of xenobiotics might release some highly reactive compounds 高活性的化合物 such as free radicals 自由基 , strong electrophiles 亲电体 or highly stressed three-member rings (epoxide 环氧化物 , azaridine 楝碱 , episufide and diazomethane 重氮甲烷 ;) which will undergo nucleophilic 亲核的 ring opening.

Freely roaming reactive compounds are not welcome in a living organism because they react randomly with macromolecules 巨大分子 , 高分子 such as DNA, RNA, and proteins. The reactions might lead to mutations 突变 or carcinogenic 致癌 changes.

Phase 1 - Biotransformation

• Includes oxidative, reductive and hydrolytic reactions.

• In these type of reactions, a polar group is either introduced or unmasked 暴露 , so the xenobiotic becomes more water-soluble and can be excreted.

• Phase I reactions are non-synthetic in nature & generally produce a more water soluble & less active metabolite 代谢物 .

• The majority of Phase I metabolites are generated by a common hydroxylating enzyme酶 system known as cytochrome P-450.

• Overall reaction is called hydroxylation 羟基化反应

RH + O2 + H2 → ROH + H2O xenobiotic (OH) hydroxyl group 羟基

polar and soluble

Figure 3.2 Hydroxylation reaction by cytochrome P-450

P-450 has a prosthetic group 辅基 inside

xenobiotic

The single-electron transfer is achieved by coupling cytochrome P-450 with another enzyme called cytochrome P-450 reductase, which has 2 prosthetic groups.

Both enzymes are embedded inside the cell into the phospholipid matrix, a component of the endoplasmic reticulum (ER), especially in the liver cells.

Phase 2 - Conjugation• The lipophilic xenobiotic is converted by phase

1 processes into polar, somewhat more hydrophilic, products may undergo further transformation into highly-soluble compound.

• These reactions involve covalent attachment of small polar endogenous molecule such as glucuronic acid, sulfate, or glycine to form water-soluble compounds.

• The final compounds have a larger molecular weight.

Two types of conjugation

1. Electrophilic 亲电子 conjugations (the conjugating agent is an electrophile)

2. Nucleophilic 亲核子 conjugations (the conjugating agent is an nucleophile)

3. Some common electrophiles are glucuronide, sulfate, acetate, glycine, glutamine, etc.

4. Only one nucleophile which is glutathione

Figure 3.10 Mechanism of conjugation of p-hydroxyacetylalanine with glucuronic acid

Glutathione 谷胱甘肽

Glutathione• is a -glutamyl-cysteinyl-glycine tripeptide,• Occurs in most tissues, but especially in the liver.• Involved enzymatic and nonenzymatic reactions.• Nonenzymatically, glutathione captures electrophiles,

acts as a low-molecular-weight scavenger of reactive electrophilic xenobiotics.

• Enzymatic reaction involving glutathione are catalyzed by a series of isozymes 同工酶 commonly known as glutathione S-transferase (GS).

• Isozymes are enzymes with different chemical compositions but performing the same catalytic functions

• The substrates include aliphatic and aromatic epoxides, aromatic and aliphatic halides.

• The conjugated product is further hydrolyzed with the removal of glutamyl and glycyl residues, followed by N-acetylation by acetyltransferase.

• The end product is mercapturic acid, which is highly water-soluble and easily excreted in urine.

Reaction between halides and glutathione

Glutathione

• Reactions of organic nitrates 硝酸盐 with glutathione.• reduction of the organic nitrate to inorganic nitrite

and oxidation of glutathione to its S-S dimer.• The nitrites formed in such reactions may interact with

amines and thus lead to the formation of carcinogenic nitroamines 亞硝胺 .

• Another reaction – by glutathione peroxidase convert reactive perioxides to alcohols, whereas glutathione is oxidized.

Reaction between organic nitro compound and glutathione

Enzyme Induction

• A phenomenon in which a xenobiotic causes in the biosynthesis of an enzyme.

• Proceeds via a cytoplasmic receptor-inducer complex, which in turn interacts with an appropriate gene to cause an in production of the enzyme.

• Cytochrome P-450 exists in different forms and these isozymes are inducible by specific agents.

Cytochrome P-450 Isozymes• The mixture of isozymes could be separated by gel

electrophoresis into distinct bands, e.g. LM2 and LM4.

• LM2 is inducible by phenobarbital 镇静安眠剂 (PB). Pretreatment with PB smooth endoplasmic reticulum and liver weight, not induce extrahepatic cytochrome P-450.

• LM4 is inducible by -naphthoflavone and 3-methylcholanthrene (3MC). Pretreatment of 3MC liver weight, slight endoplasmic reticulum, hepatic and extrahepatic P-450 enzymes.

Inducers of Cytochrome P-450

P-450 inhibitors• Reversible or irreversible.• Reversible inhibitors

– Slowly metabolized substrate of P-450– Occupy the active site of the enzyme and thus retard the

processing of other xenobiotics– E.g. 2-diethylaminoethyl 2,2-diphenylvalerate

Irreversible P-450 Inhibitors 抗化剂• An examples is carbon tetrachloride

– Causing peroxidation of lipids– In turn destroys cell membrane integrity– Leads to a subsequent loss of P-450

• Assessment of inhibition effect– Measuring an increase in sleeping time of animals

anesthetized with hexobarbital 环己烯巴比妥 – As hexobarbital is inactivated by cytochrome P-450,

inhibitors of P-450 prolong sleeping time, whereas inducers shorten it.

Environmental Inducers of P-450

• Insecticide DDT (1,1,1-trichloro-2,3-bis(-chlorophenyl)ethane)

– Induction of P-450– Reduced the number of mammary tumors produced

by dimethylbenzanthracene, because of the production of noncarcinogenic hydroxylation of dimethylbenzanthracene.

– Induction of epoxide hydrolase, glutathione S-transferase.

Environmental Inducers of P-450

• Other chlorinated hydrocarbon pesticides e.g. aldrin, dieldrin, hexachlorobenzene and hexachlorohexane.

• Polychlorinated biphenyls (PCBs)– Used as insulating fluids in capacitors, transformers,

vacuum pumps and gas transmission turbines.– Induction of -nitrophenol and testosterone glucuronyl

transferases– Increase liver weight and microsomal protein.

Environmental Inducers of P-450

• TCDD (2,3,7,8-tetrachlorodibenzo--dioxin)– Very potent inducer– Formed on incineration of chlorinated organic

substances, found in exhaust and in ash from municipal incinerators.

– Also formed in the process of pulp bleaching in paper manufacturing

– A by-product of the manufacturing of a herbicide and a wood preservative.

Structures of DDT, biphenyl and tetrachlorodibenzo--dioxin 二恶英

Inducers for phase 2 metabolizing enzymes

• Found in cruciferous vegetables e.g. broccoli 绿花椰菜 , cauliflower 花椰菜 , other cabbage-related plant 甘蓝 ; 卷心菜 .

• Induce glutathione S-transferases and quinone reductase

• A diet rich in green and yellow vegetables lowers the risk of cancer in human

Activation of precarcinogens 前致癌物

• In some cases the metabolism of xenobiotics leads to the formation of unstable intermediates that react with cellular macromolecules.

• The reaction leads to mutagenic or carcinogenic transformation.

Precarcinogens

• 2-Naphthylamine 萘

– Used in dye 染料 manufacturing– Produce bladder cancer among workers– Becomes a carcinogen upon N-hydroxylation by

cytochrome P-450– The Hydroxylamine is stabilized by glucuronidation, and

becomes harmless– Hydrolyzed back to carcinogenic hydroxylamine by -

glucuronidase in the kidney or by acidic pH in the urine.

Carcinogenic activation of 2-naphthylamine

carcinogen

carcinogen

harmless

Precarcinogens

• Dichloroethane– Waste product of vinyl chloride production and also a

laboratory solvent– Its analog, dichloromethane, is used as a gasoline additive

and as an insecticide– Carcinogen and mutagens– Conjugate with glutathione to form haloethyl-S-

glutathione which is structurally similar to sulfur mustard, used as war gas during world war I

– Haloethyl-S-glutathione spontaneously forms an unstable three-member ring that, upon ring opening, reacts with cellular macromolecules.

Carcinogenic activation of 1.2-dichloroethane

Precarcinogens

• Vinyl chloride– Starting materials in the manufacture of poly(vinyl

chloride) plastics– Associated with high frequency of angiosarcoma,

a rare liver cancer.– Carcinogenic activation Involves epoxide

formation

Carcinogenic activation and further metabolism of vinyl chloride

Precarcinogens

• Aflatoxins 黄曲霉毒素– Produced by a mold, Aspergillus flavus– Contaminates crops such as corn and peanuts– Aflatoxin B1 : can be metabolized by cytochrome

P-450 to a carcinogen

Carcinogenic activation of aflatoxin B1

Precarcinogens

• Benzo[]pyrene– A major polycyclic hydrocarbon carcinogen in the

environment– Occurs in industrial smoke, cigarette smoke and

tar, and in fried, broiled, or smoked food.– Metabolized by P-450 to form an epoxides, 7,8-

dihydrodiol-9,10-trans-epoxide that is a carcinogen with half-life of 8 min. It is probably long enough to react with DNA.

Carcinogenic activation of benzo[]pyrene

Precarcinogens

• Nitrosamines 亚硝胺 – Formed by the reaction of nitrite ions with

secondary and tertiary amines, e.g. dimethylamine used in rubber, leather and soap manufacturing

– The formation of carcinogenic nitrosamines can be prevented by compounds that compete with secondary and tertiary amines for the nitrite ion, e.g. primary amines, ascorbic acid and tocopherol.

Carcinogenic activation of dimethylamine by reaction with nitrite

ions