---start biochem 11.25.96----- handout: CHEMICAL CARCINOGENS AND ANTICANCER AGENTS - note page numbers are wrong, 10 should be 12, 12 should be 11, 11 should be 10. LECTURE OUTLINE: 1: environmental and industrial chemicals, DNA and RNA transforming viruses, radiation, and genetic inherited factors = main causes of cancer. 2. classification of chemical carcinogens: industrial/environmental pollutants, synthetic compounds, food constituents and food additives. 3. the role of cytochrome P450 monooxygenases in hte metabolism of physiologically important lipids and also metabolic activation of chemical carcinogens. 4. chemical carcinogenesis as a multistep process requiring the action of both tumor initiators (chemical carcinogens) and tumor promoters (mitogens or cell cycle regulators) 5. mechanisms of action and source of anticancer agents 6. critical targets of chemical carcinogens: dominant and recessive oncogenes and their role in the neoplastic outcome. -- MAJOR CAUSES OF CANCER: chemical carcinogens: >90% DNA and RNA tumor viruses: a few years ago people thought these were very important in causing cancer, but now most people believe it isn't a major factor, though many viruses CAN transform cells in vitro and in vivo. GENETIC predisposition: some of the genes that are inherited, esp tumor suppressor genes, esp in mammary, colon cancer, are inherited in mutated form leaving the individual more vulnerable. RADIATION exposure: quite common, esp today. Hormones: as discussed by doc shapiro. We're going to concentrate here mainly on the chemical carcinogens. on p 3 of handout the lecture is basically summarized. Critical steps in activation and inhibition of carcinogenesis figure....note that carcinogenesis is a multistep process. one or two cells are altered, then they keep dividing... starting with CCs (Chemical carcinogen), which are essentially inactive as they exist in nature....they are quite lipophilic. they are activated invariably in the body, usually in the liver although other tissues can also participate. in the activation process cytochrome P450 system is invovled. cyt p450 has a heme prosthetic group. so when it is reduced w/carbon monoxide it shows an absorption of 450 and that's why it is named that. these enzymes belong to a multigene family - well over 200 genes are involved. so there are a large number of genes for different isoforms of the enzyme. along with a helper protein, cyt p450 reductase, and reduced NADPH to function as the electron donor, you activate the CCs into UCs (ultimate carcinogens), then, if they aren't stopped by tumor inhibitors, the UCs to bind to DNA and induce mutations (induction of cancer). then tumor promotors cause induction of cell division. so, after the mutations are induced, you're still fine. the cells aren't yet dividing, so it is no problem if they carry mutations. but if you are exposed to tumor promotors, which invariably induce cell cycle or move cells from g0 to s phase, then you have induced cell division and the mutant cells divide and increase their number, and you have a hyperplastic nodule, the first visible sign of cancer formation. but these nodules are still pretty benign and treatable by sx or radiation. but once the nodule grows, a number of steps occur and they become neoplastic and start migrating and metastasizing. in between hyperplast and neoplasia you can inhibit the transformation by retinoic acid, dialyl sulfide, and perillyl alcohol - because these compounds turn them back into near normal cells. types of chemicals involved: Tumor initiators: chemical carcinogens, when activated by class I drug metabolizing enzymes induce mutations and tumor initiation. these chemicals are very lipophilic and are activated by cytochrome p450 enzyme system. depending on the structure of the chemical there are many isoforms of the activating enzyme. tumor promoters: chemicals which induce cell proliferation and amplify the mutational damage, or help populate damaged cells. tumor inhibitors: chemicals that detox activated carcinogens by inducing class II drug metabolizing enzymes, and chemcals which cause tumor regression by altering transcription regulation and dedifferentiation. NATURE AND SOURCE OF CHEMICAL CARCINOGENS: there are tons of potential carcinogens. most of them only induce cancer in extremely high doses. others are definitely dangerous. I industrial/environmental pollutants the largest class here is polycyclic aromatic hydrocarbons: PAHs. these compounds have a number of fused benzene ring structures. these are highly insoluble and highly potent carcinogens. benzanthracene can be activated in lung and other tissues, and is one of the most potent carcinogens ever known. it causes multisite lung, skin and other tissue cancer. chemicals in this class are usually inhaled, part of auto exhaust, cigarette smoke, or even in the kitchen. these result from incomplete combustion of organic matter. the subclass of this, AROMATIC AMINES and some azo compounds eg dimethyl amino azo benzene, are very colorful compounds, used a lot in oil in paint. so they are common inducstrial byproducts. aromatic amines cause bladder and gi cancer, and the DAAB causes liver cancer too. II industrial/synthetic eg pesticides these are collectively classified as polychorinated biphenyls, PCBs. these are a major industrial byproduct. since 1979 use has been banned, so they have been buried. important property of these: extremely stable, resistant to heat, acid, alkali, fire. 10-15 yrs ago compounds of this type were used as fire repellents, furniture finishes, paint, to reduce fire hazard. now we know they are very dangerous. were also used to make electrical capacitors, carbonless paper for credit cards etc. another common use was as pesticides and defoliating agents, esp the chlorinated dibenzo p-dioxin (TCDD) was used in viet nam, and DDT was used in many parts of the world to control malaria - but it seems to have killed lots of birds :(. and now many wells are contaminated and areas are contaminated. and the mosquitoes are resistant to the DDT now anyway. many of the lands sprayed 15-20 yrs ago are still contaminated. and if animals eat the grass, whoever eats the animal gets a dose of it. this stuff is very inert and the metazooan system can't metabolize it or convert it to an excretable form. if you get some, it accumulates in your skin, liver, brain, etc. it's probably also a tumor promoter as well as a carcinogen. these are not modified by the p450 system! they are already able to induce cancer. they may be modifying some other enzyme causing it to cause the mutations. there are about 400-500 kinds of these buried underground and we don't know how to get rid of them. III food contaminants and additives: plant and fungal products: i) aflatoxin B1 - a product of aspergillus flavus mold which grows at about 30-35 deg C and 80-95% humidity. so it's pretty common. this is a VERY dangerous hepatotoxin and hepatocarcinogen. this compound was discovered within vet med. about 40 yrs ago they found suddenly hundreds of thousands of turkeys died. they turned out to have badly damaged livers. then some horses died. now we knwo it was a result of contaminated feed that had this toxin in it. cows, dogs, cats, people are very sensitive to this compound. sheep are very resistant and so are mice, and so are monkeys, to a degree. this is due to glutathione s transferase system. mice have a ton of this enzyme, very high level. even marine species are very sensitive to this stuff. this compound induces liver dz, liver toxicity, usually in africa, parts of asia, south america, was common, farmers saw animals die and weren't sure why, but their liveers had massive lipid inclusion. it depends on how much they've eaten. massive dose: lipid buildup, cirrhosis and death ensue rapidly. if eaten in subtoxic amounts over longer time will cause cancer. ii) pyrolizidine alkaloids; plant toxin, horses very sensitive. herbal medicines may contain these and cause liver cancers in those who take those preparations. iii) safrole (essential oils of many spices eg ginger, pepper, nutmeg, turmeric, cinnamon, mace, etc) amount in the spices is so small, it's not possible to get toxic dose that way iv) cycasin: glycan type of compound, very strange compound, just mentioning it b/c it's important in other parts of the world eg guam, okinawa - the seed of a fruit? it's poisonous. very sweet seed, meal is made from it and everyone likes it. but once you ingest it, there is a conversion into an azo type compound via the cyt p450 path and it becomes dangerous. NICOTINE: by itself is inert. during tobacco curing as well as in the lung tissue it is converted into a nitrosamine which is quite dangerous and highly carcinogenic. causes most of smoker lung epithelial cancer. it's hard to separate out cancer it causes from cancer caused by other carcinogens in smoke, though. food additives i) nitrates, nitrites, nitrosamines: can be converted in saliva, bladder, into dangerous nitrosamines which cause colon cancer and GI cancer. in fact, most colon cancer and GI cancer is caused by these. they're in processed and smoked and salt cured food: anchovies, etc. ii)aromatic amines: food coloring iii) organochlorides: pesticides iv)arylonitriles: didn't go over v)vinyl chloride: food packaging/plastic. REMEMBER: PAHs.: daily exposure aflatoxin B1: very dangerous hepatotoxin/carcinogen nitrosamines: how nitrates and nitrites become nitrosamines in the body, and also they are formed during food processing. Ok. so - these have to be metabolized. so they cytochromeP450 enzyme family - a huge family - many genes....remember it's not just that they function to activate carcinogens. it was likely evolved to take care of cholesterol. p450 enzymes are as ancient as cholesterol. the body then figured it was also a good way to metabolize (eg, convert, modify) other, foreign substances as well. functions of P450 enzymes 1. activation and inactivation of drugs: apirin, tylenol, erythromycin, warfarin, debrisoquine, halothane and other anesthetics 2. chemical carcinogenesis: activation of xenobiotic (foreign) chemicals and carcinogens into DNA reactive forms 3. participation in physiological pathways: steroid hormone biosynthesis and cholesterol metabolism, fatty acid metabolism and prostaglandin biosynthesis. realize there are special forms of CytP450 for different functions. Inducers of cyt p450 steroid hormones, parathyroid and growth hormones. alcohol, barbiturates eg phenobarbitol, and number of drugs. many carcinogens such as polycyclic aromatic hydrocarbons, dioxins (TCDD) and aromatic amines and some alkaloids. so p450 isn't jsut a bad guy. also important for metabolic functions in 3 above. also note that the inducers may induce particular isoforms of the cyt p450 - not all the same form. alcohol induces a couple, steroids induce a different one, etc. note that PAHs and TCDD have special cytosolic receptors which, when activated, go into nucleus and induce transcription. ---break--- so we talked about the inducibility of cyt P450- that means that these are induced transcriptionally and a large amount of protein is then made P450 is localized mostly in the ER and also in the mitochondria as well. it's mostly in the liver, also in some other tissues eg kidney brain lung intestine. it's NOT found in RBCs and skeletal muscles. all tissues have different isoforms. the P450 in fig 23.6 is in ER. note the p450 is embedded in the membrane and has a transmembrane domain not seen in the diagram. the p450 reductase needed to transfer e- from the NADPH to the p450 is also associated with the membrane, bound to it by its hydrophobic tail. the p450 has a substrate binding pocket. the reductase functions only to transfer electrons. p450 actually catalyzes the reaction (whatever it is). FAD and FMN (flavin adenine dinucleotide and flavin mononucleotide) are present in the reductase- they are nucleotide cofactors and they help hold the electrons or something - they need to hold the two electrons that come from NADPH and then the electrons are transfered one at a time to the heme group of the P450 itself. the actual reaction: (SuH = reduced substrate) NADPH + H+ +O2 + SuH----cyt p450 and cyt p450 reductase--->NADP+ + H2O + SuOH O2 + 2 e-----> O* + O* note that only the reduced ferrous can bind molecular oxygen. also note that the oxygen becomes two reactive oxygen species - one of which is added to substrate, and one of which is added to hydrogen to make water. now, alcohol induces cyt P 450, and that particular isoform is particularly likely to keep accepting electrons into its heme pockets and making free radicals, and then if there's no substrate, you have these reactive species floating around, it's not very good for you. be sure to understand that the P450 is TOTALLY responsible for catalytic process of above rxn. the reductase is ONLY an electron carrier. p9 of handout: activation of carcinogens by cytp450 monoxygenase eg, aflatoxin b1 is converted to an epoxide by an isoform of p450. the epoxide is very reactive, electron deficient compound. . remember that different isoenzymes are invovled for different carcinogens, that they create electron deficient groups that are highly reactive, and hwich then bind to e- rich groups such as those found in AAs, nucleotides, phosphodiester bridges of DNA P450 enzyme levels modulate the pharmacological effectiveness of mny drugs Drbrisoquine, an antihypertensive, is metabolized by P4502D6 which is induced by barbiturates. Warfarin: an anticoagulant, is metabolized by P4502A1/2A2 which is induced by barbiturates and sedatives. synthetic estrogens/progestins (oral contraceptives) are metabolized by P4503A6, which is induced by the antibiotic rifamycin. so, if you give one of these drugs to an animal which is also receiving an inducer of the p450 isoform which inactivates the first drug, you're not doing anything useful here. just understand that the P450 and P450 reductase are transmembrane proteins, that the heme is in the P450, and the e- and handed off to the P450 by the reductase. again, multiple forms of P450 act on different substances, converting them to "electrofiles" which then go on to become conjugated and excreted (GSH) or to develop mutations from reactive oxygen species and possibly become cancerous. TUMOR PROMOTERS: those that induce proliferation , move cells into S phase from quiescent phase, g0 non dividing phase, into S phase. the Phorbol ester is a powerful tumor promotor isolated from seeds of croton tiglium plant of se asia. it induces protein kinase c- activating g protein part (receptor ligand interaction). this ester induces the protein kinase c pathway which in turn phosphorylates other proteins, forcing the cells to proliferate (i couldn't understand his accent here so i missed some stuff). even in isolated cell culture if you add this compound you induce protein kinase c and they go into seoiisaojwoierpoiiofoieroe and they go into worpwe condition (???) other tumor promotors: phenobarbitol and other barbs. alcohol, cigarette smoke, dioxins, asbestos, saccharin and cyclamate. not 100% sure about saccharin and cyclamate. what a tumor promoter does... experiment: formation of skin papillomas in mice. a number of carcinogens were given. if you give the initiator and wait for a long time, you still don't see cancer. you have to give the initiator and also give a promotor. even if you give one dose of initiator, then wait a year, then give the promoter, you get a high incidence of the tumor. these tumors start as a few cells, but then grow, because the cells are quiescent until the promoter induces cell proliferation. now, if you give a promoter, and wait until it wears off to give the initiator, you don't get tumors. you have to give the initiator first, then the promoter. carcinogenesis IS a multistep process. a slide is shown of colorectal cancer in man. it looks like a very warty mucosa...some small clumps of tissue, some bigger.kind of cauliflower like. start as adenomas, end up as carcinomas. only the carcinoma stage is dangerous because only then does it invade through the basement membrane. note that once the tumor gets big enough, cells on the inside start having problems getting nutrients and develop hypoxia. then there is a high risk of free radical formation, and that causes the tumor to progress even more. what about tumor inhibitors? two types of chemicals...the detoxifiers are one - they physically conjugate and eliminate the activated carcinogens. Glutathione system: glutathione s-transferase glutathione + activated carcinogen---glutathione s-transferase--->glutathion conjugate GLUCURONIC ACID SYSTEM UDP glucuronic acid + activated carcinogen---UDP glucoronase **missed end of rxn :( also antioxidants, vit C and E, neutralize e- deficient groups, butylated hydroxy anisol: induce class II enzymes like glutathione s transferase aromatic isothiocyanates from cruciferous veggies also induce class II enzymes, eg phenethyl isothiocyanate is a lung CA inhibitor flavones also induce class IIs. found in fruits and veggies. chemicals that cause tumor regression: diallyl sulfide and related compounds from garlic and broccolli are effective against liver tumor and also affect growth patterns of colon, lung, and other cancers. retinoic acid and related retinols: produces beta carotene, from dark green and yellow veggies. effective against may epi cancers, eg lung, buccal, stomach, mammary, vaginal, etc. these compounds bind to retinoic acid receptors (something about steroid receptors...not sure. definitely distinct from steroid receptors, though) that are transcription factors, and they induce or repress transcription. they also modify many proteins in the cytoplasmic and nuclear compartments. the mechanism has been worked out pretty well. Extracts of orange peel (limonene) and lavendar(perillyl alcohol) also have anticancer activity. ----end----