326

Tamour progression to maiig- nanny is often associated with gross structural and numerical chromo- somal abnormalities. However, it is believed that this massive destabilization of the genome without clonal origin is not the prerequisite for efficient cell pro- liferation.

Need for mechanistic distinction The distinction between geno-

toxic and epigenetic activity of carcinogens (Fig. 1) might seem rather theoretical for the cancer victim. However, we consider it essential to understand the bio- logical processes underlying the process of carcinogenesis, since it is an important aspect when data obtained from animals are to be extrapolated to humans. For exam- ple, when carcinogens are found to be transformed into chemically reactive metabolic intermediates interacting with DNA in some ani- mal species, it will be important to find out whether the compound is metabolized in a similar way in humans. Additionally, the dose- response relationship might be different for genotoxic carcinogens and those carcinogens that act only at high dose levels by inducing cytotoxicity and regenerating cell division, or those non-mutagenic carcinogens that act at hormonal levels35.

An understanding of the mechanisms of carcinogenic activ- ity might allow for the develop- ment of a battery of short-term tests able to detect chemicals active in defined steps of carcinogenesis. This will facilitate better risk eval- uation of chemical carcinogens. New tests to be devised should permit a comparative analysis of different types of mutations and the detection of chemicals that stim- ulate cell proliferation and inter- fere with cell differentiation.

References 1 Bishop, J. M. (1987) Science 235,305-311 2 de Kiein, A. (1987) Mutat. Res. 186,161-

172 3 Klein, G. (1987) Science 238, 1539-1545 4 Cavenee, W. K., Koufos, A. and Hansen,

M. F. (1986) Mutat. Res. 168,3-14 5 Cavenee, W. K. et nl. (1983) Nature 305,

7?&,11d ,*, ,- 6 Armit+!, P. (1%) %_jiioit. iiealth Pers-

pect. 63, 195-201 7 Land, H., Parada, L. F. and Weinberg,

R. A. (1983) Science 222,771-778 8 Zarbl, H., Sukumar, S., Arthur, A. V.,

Martin-Zanca, D. and Barbacid, M. (1985) Nature 315,382-385

9 Quintanilla, M., Brown, K., Ramsden,

M. and &hUiA, A. (1986) Nature 322, 7tua-l .- __

10 Lutz, W. I:. (1979) Mutut. Res. 65,289-356 11 Shay, J. W. and Werbin, H. (1987) Mutat.

Res. 186,149-160 12 Cerutti, P. A. (1985) Science 227,375-381 13 Kunz, B. A. (1982) Environ. Mutagen. 4,

695-725 14 Brusick, D. J. (1987) Mutat. RCS. 189,1-6 15 Randerath, K., Reddy, M. V. and Diaher,

R. M. (1986) Carcinogenesis 7, 1615-1617 16 Reynolds, S. H., Stowers, S. J., Maron-

pot, R. R., Anderson, M. W. and Aaron- son, S. A. (1986) Proc. Natl Acad. Sci. USA 83,33-37

17 Berenblum. I. 11941) Cancer Res. X44-48 18 Biisser, M.’ T.’ and Lutz, W. K.. (1987)

Carcinozenesis 8, 1433-1437 19 SchulteIHermann, R., Timmermann-

Trosiener, 1. and Schuppler, J. (1983) Cancer Res. 43, 839-844

20 Goustin, A. S., Leof, E. 8.. Shipley,G. D. and Moses, H. L. (1986) Cancer Res. 46, 1015-1029

21 Spom, M. B. and Roberts, A. 8. (1985) Nnture 313,745-747

22 Moser, G. and Maier, P. (1987) Eur. 1. Cell Biol. 44,156-160

23 Fisher, P. B., Miranda, A. F., Babiss, L. E., Pestka, S. and Weinstein, I. 8. (1983) Proc. Natl Acad. Sci. USA 80,2961- 2965

24 Murray, A. and Fitzgerald, D. (1979)

TIPS - September 1988 [Vol. 91

Biochem. Biophys. Res. Commun. 91,39S- 401

25 Pardee. A. 8. (1987) Cancer Res. 47,1488- 1491

26 Holzer, C., Maier, P. and Zbinden, G. (1986) Exv. Cell. Biof. 54, 237-244

27 &n&h, i&‘., Lauer, B., Timmermann- Trosiener, I., Barthel, G., Schuppier, J. and Schulte-Hermann, R. (19%~ Car- cinogenesis 5, 4S3-458

28 Holzer, C. and Maier, P. (1987) Toxicol. In Vitro 1,203-213

29 Schwarze, P. E., Pettersen, E. O., Shoaib, M. C. and Seglen, P. 0. (1984) Curcino- genesis 5,1267-1275

30 Solt, D. and Farber, E. (1976) Nature 263, 701-703

31 Dzarlieva-Petrusevska, R. T. and Fusen- ing, N. E. (1965) Carcinogenesis 6, 1447- 1456

32 ;;;boim, H. C. (1982) Science 215,1247-

33 Fahrig, R. (1984) Mol. Gen. Genet. 194, 7-14

34 Pelling, J, C., Neades, R. and Straw- hecker, J. (1988) Carcinogenesis 9,66!%67

35 Lutz, W. K. (1986) J. Cancer Res. Cfin. Oncol. 112,85-91

TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin DDT: l,l,l-trichloro-2,2-bis(p-chloro-phe-

nyl)ethane

James Shepherd and Christopher J. Packard

Several recent clinical trials have demonstrated that strategies that lower plasma cholesterol levels do indeed have a beneficial effect on events related to coronary heart disease. A major strategy to lower cholesterol levels is via pharmacological intervention, James Shepherd and Christopher Packard evaluate the mechanisms and effectiveness of currently available classes of drug that act by lowering stero: absorption, lowering sterol synthesis, or interrupting the enterohepatic circulation.

Health care services in industrial- ized countries are becoming so bogged down in the treatment of established disease, that little is left to offer the population by way of preventive therapy. As a conse- quence, chronic degenerative ill- nesses are placing an ever increas- ing burden on the resources of primary health care systems, des- pite accumulating evidence that some of them may be improved by simple measures aimed at lifestyle modification.

lames Shepherd is Professor and Christopher]. Packard is Principal Biochemist in the Depart- ment of Biochemistry, Royal Infir&y, Glasgow G4 OSF, UK.

Coronary artery disease, for example, which is responsible for about a third of all deaths in industrial societies, appears to respond to interventions designed to alter pop~lafion attitudes and habits. Several major risk factors - hypercholesterolaemia, hyperten- sion and cigarette smoking - con- tribute to its development, and when treated produce significant advantage for the individual. The Lipid Research Clinks Coronary Primary Prevention TrialI, for example, offered convincing evi- dence that lowering plasma chol- esterol levels in asymptomatic hypercholesterolaemic men reduces their risk of having a myocardial

@I 1989. Elsevier Publications, Cambridge 0165 - 6147/g8/Wk!l@

TIE5 - September 1988 fVd. 91

1

ENTEROHEPATIC ClRCULATlON

Fig. 1. ~~ula~~ steps fft choiestem/ metabc&m, ?he nf;g &&&&~I$ e$fnr f&e! we ingest each day is hydro!ysed in the gut, absorbed and packaged info chyiomicmns for dgriveqr to thff Nver sterof pocl. This pcol is also fed by choleslerol synthesis in the liver itself. Three key regu/atofysites for cholesterol metabclism are: intestinalabsorption (1); hepatic synthesis (2); and bi!iav secretion (3).

infarction. A rapid succession of additional reportsz4, culminating in the eager1 awaited Helsinki Heart Stud s underscored the value of chol&teroll reduction in any program designed to attack coronary heart disease.

End-points From the above clinical trials, it

was clear that only major end- points, such as death or new myocardial infarction,, could be used with any confidence. The low frequency6 of fatal coronary events even within high-risk ~pulations (- 0.3% per annum) made such studies both prolonged and expen- sive. But we know’ that ischaemic heart disease mortality is directly related to the progression of coron- ary atherosclerosis, a phenomenon which is amenable to direct visual- ization by contrast radiography.

This approach is to some extent limited by the invasiveness of the ~athete~zation procedure itself and by the technical probkzms inherent in quantifying the degree of arterial stenosis. Moreover, since exposure of apparently healthy individu~$ to the hazards of repeat angiography is ethically unacceptable, prospective studies of this kind are usually limited to examination of diseased indi- viduals who are not necessarily ~p~sentative of the population as a whole,

It was therefore gratifying to

learn from the recently published Cholesterol Lowering Atheroscler- osis Study4 that the benefits of plasma cholesterol lowering are measurable net only in terms of coronary event reduction, but also in relation to Coronary occlusion regression.

The pharmaceutical industry has not been slow to realize the significance of these findings. New lipid-lowering agents and old friends in fresh guise are being offered to the clinician. This development certainly widens choice but with it carries the need to establish the risk/benefit ratio of the regimen which may need to be prescribed for the remainder of the patient’s life.

The in~oduction of a new agent to the market implies that the manufacturer is able to substan- tiate its efficacy, but often the detailed mechanism(s) respons- ible for its actions are incompletely understood. This has certainly been true until recently as far as cholesterol lowering drugs are concerned, but as our knowledge of sterol metabolism develops so does our appreciation of the key regulatory steps responsible for its regulation. This review outlines the main pathways of sterol flux through the hepatic and systemic circulations and indicates those sites where pha~acological inter- vention may lead to effective plasma cholesterol reduction.

327

Sterolabsorptioninhibitors There are at least three indentifi-

able stages involved in the absorp- tion of cholesterol from the intesti- nal lumen (Fig. 1). Firstly, sterol esters are hydrolysed and the liberated cholesterol solubilized in mixed micelles by interaction with bile acids and luminal phospho- lipid. These particles cross the unstirred water layer to sites on the enterocyte mucosal surface that are responsible for sterol absorption,

Within the cell the choleste31 is reesterified via the agency of acyl COA : cholesterol acyltransferase (ACAT) then packaged into chylo- micron particles which are released in a wave into the plasma. There, lipoprotein lipase is responsible for hy~olysing the core triglyceride of these particles, producing remnants that retain most of the cholesteryl ester. Rapid receptor-mediated uptake of the remnants results ;n deposition of the sterol ester within hepatocytes.

The antibiotic neomycin has long been known to interrupt chol- esterol absorptior8’. It appears to do this by precipitating micellar sterol in the gut, thereby prevent- ing its absorption by the mucosa. Consequently, faecal cholesterol excretion is markedly enhanced9 and chylomicron-mediated deliv- ery of sterol to the liver reduced. H~er~oleste~laemic patients treated with neomycin show a reduction of about 25% in plas- ma cholesterolg~‘*. Interestingly, recent studies have shown that, contrary to expectation, the fall in circulating low-density lipoprotein (LDL), the major sterol transporter in the plasma, does not come pri- marily from an increase in its clear- ance from the plasma but rather from a reduction in synthesis*‘.

This picture confirms the events that we described following chol- esters1 feeding12 and indicates that hepatic sterol metabolism is more compks than first e~v~~ged. Such evidence suggests that there must be several distinct regulatory metabolic pools of stem1 within the hepatocytes13. One is presum- ably associated primarily with endogeno~s lipoprotein prod- uction while another seems to be fed by LDL catabolism and is more involved in regulating the activity of the LDL receptor.

Despite the significant hypo- cholesterolaemic action Of neo- mycin, most clinicians would be

328

assimilation -

Fig. 2. Actions of acyi CoA rctmleeteml acyl&ransferase inttibitots. These my itwrease free st8rotaveilabitity for removal via HDL, Suppress endOgenOUS St8tvtSynthaSiS, an&or suppress LDL receptor activity. 0, Cnalesterot.

wary of using the drug as a long- term choleste~l-lowe~ng agent because it confers a substantial risk of ototoxicity.

The intestinal mucosa is limited in the number of sites available for sterol absorption. Consequently, when plant sterols are given in -high dose fl@-15g per day) they are able to block by competition the uptake of cholesterol without themselves being absorbed in the process. Alternatively, they may act by limiting the solubility of cholesterol in intestinal mixed micelies. Patients receiving such therapy show variable reductions in plasma cholesterol’4J5 presum- ably via a mechanism similar to that triggered by neomycin therapy.

The efficient ~sim~ation of chol- esterol by the gut requires that it be esterified within the mucosal cell by the microsomal enzyme ACAT. Several agents (cetaben, compound 58-035, etc.) have been developed specifically to block this reaction in the hope that sterol absorption might be inte~pted16, As yet, the clinical efficacy of such drugs remains unproven since they do not uniformly produce decrements in plasma cholesterol. However, their action is not neces- sarily limited to the intestinal mucosa and so measurement of plasma cholesterol levels might not be an appropriate indicator of their therapeutic potentiall’. Their local actions on arterial intima, for example, may prevent intracellular accumulation of choleste~l esters even in situations where plasma sterol levels remain high (Fig. 2).

This is exemplified in a recent study in which chlorpromazine

was used to inhibit arterial ACAT activity in cholesterol-fed rabbits. Treatment with the drug reduced aortic ACAT levels by about 40% without inducing any major chiinges in circulating lipoprotein levels. However, significant sup- pression of aortic cholesterol accu- mulation did occur, consistent with the view that agents such as chlorpromazine limit the develop- ment and progression of athero- sclerosis. Proof of such benefit could only be obtained by under- taking prospective clinical trials or angiographic studies in humans, an expensive and daunting pro- cedure. So far, studies of this kind have not been approved and the ACAT inhibitors remain experi- mental.

Sterol synthesis ~~ibi~~ Aimost a decade ago, several

alkaloids isolated from Penicillium citrinum were found to be owerful competitive inhibitors s of 3- hydroxy-3-methylglutaryl coen- zyme A reductase (HMG-CoA reductase), the rate-limiting enzyme of cholesterol synthesis (see Ref. 19). Part of these mol- ecules mimics almost exactly the natural substrate of the enzyme (Fig. 3) so that they are able to exert their actions when present in very low concentration. If administered to certain animals (e.g. mice, hamsters or rats) they produce negligible changes in plasma chol- esterol, but their effectiveness in higher species like dogs, monkey and humans is well proven . Indeed, they are now among the most potent compounds available for the control of human choles- terol levels.

TlPS - September 1988 IVol. 91

Metabolic studies have shown that by inhibiting cholcsterolo- genesis in the liver these drugs activate LDL receptors which, in turn, promote assimilation of the lipoprotein from the plasmazl. Long term clinical experience of their utility is limited but so far clinical opinion in the United States and Sweden, where they are available on prescription, suggests that they are uniformly effective and produce minimal side-effects. Agents of this kind are not yet approved for use in most other countries.

Despite the fact that clofibrate has been available for almost 30 years, there is still some doubt as to its precise mechanism of action. However, recent studies of more potent s~ond-gen~ation deriva- tives (like fenofibrate and bezafi- brate) have revealed (Fig. 4) that they act largely by lowering plasma triglyceride by reducing the circulating level of very-low- density lipoprotein (VLDL).

Kinetic studies in humans have shown that they achieve this by a combination of effects. They reduce VLDL synthesis”* possibly by limiting fatty acid substrate availability. However, their main action seems to be to increase eatabolismes of these particles by stimulating lipoprotein lipase which is the enzyme responsible for the bulk of triglyceride removal from the plasma. It depletes the VLDL particle of its core triglycer- ide, causing it to become smaller and enriched in cholesterol and protein.

The end product of lipolysis, LDL, is susceptible to receptor- mediated clearance from the plasma, a process which is again

OH

H,C

HO*H

3.hyd~xy-3-methyl glutarate

F&j. 3. Sfruchms of pravastatin fan inhibitor of tftUG4M reduetas8) and 3-h~~~/-3-me~y/~/utarat8.

TIPS - September 1988 [Vol. 91

1 Lipid Research Clinics Program (1984) 1. Am. Med. Assoc. 251,351-364

2 Nikkila, E. A., Viikinkoski, P., Valle, M. and Frick, M. H. (1984) Br. Med. J. 289,

.220-223 3 Amtzenius. A. C. et al. (1985) N. Engl. I.

Med. 312,805-811 4 Btankenhdm, D. Ii., Nesaini, S. A.,

Johnson, R. L., Sanmarco, M. E., Azen, S. P. and Caskin-HemphiB, L. (1987) J. Am. Med. Assoc. 25?,3233-3240

5 Frick, M. H. et al. (1987) N. Engl. 1. Med. 317,l237-1245

6 MultipIe Risk Factor Intervention Trial (1982) J. Am. Med. Assoc. 248,1465-1482

7 Bruschke, V. V. D., Wijen, T. S., Kolstera, W. and Landmann. J. (1981) Circulation 63,527-536

8 Samuel, P. (1979) N. Engl. J. Med. 302, 595-597

lcbrates on @oproto metabolsfn,

stimulated by fibrate therapy. The mechanism here is not completely understood but seems to involve activation of the LDL receptor pathwag4. Although fibrates are clearly not competitive inhibitors of HMG-CoA reductase, fibrate treatment does inhibit the activity of this enzyme in rat liver or in human peripheral blood mono- cytes.

This led us to study the effects of such compounds on LDL metab- olism in both h~e~oles~e~l- aemic24 and hypertriglyceridaemic subjects25. When patients are given fibrates the LDL receptor pathway is activated so that more ~oles~eml is cleared from the plasma by this route (Fig. 4). Consequently, as was shown by fenofibrate ad- ministration, less sterol is available for channelling into potentially atherogenic receptor-independent pathways25. Although this mech- anism seems to be common to all fibrates, we still do not understand how they inhibit HMG-CoA re- ductase at the molecular level.

Bile acid synthesis The liver handles gram quanti-

ties of bile acids each day en route for the intestine. There they are reabsorbed and returned via the portal vein in the enterohepatic circulation. The system is, however, not entirely efficient since approximately 15% is lost during the cycle. This shortfall is replaced by chole&erol oxidation within the hepatocyte in a se- quence of reactions rate-limited by the enzyme cholesterol 7ar- hyd~xylase, which is normally

suppressed by bile acids returning to the liver.

Interruption of the enterohep- atic circulation either by surgery (terminal ileal bypass) or by insoluble bile acid binding drugs like coIestyramine or colestipol produces a substantial decrement in the plasma cholesterol of virtu- diy all subjects”. One exception, noted several years ago, is the rare patient who is homozygous for familial hypercholesterolaemia, and who car be induced to excrete grams of bile acids each day by this procedure without showing any change in plasma cholestero126,

The explanation for these find- ings lies in the key observation that the LDL receptor constitutes the critical link between plasma and intracellul~ sterol pools. Sequestrant resin therapy, by acti- vating cholesterol 7a+hydroxylase, drains an inarahepatic regulatory sterol pool, activates hepatocyte LDL receptors and lextracts LDL cholesterol from the circulation*‘. The potential hypocholesterol- aemic action of these compounds is not fully realized since their effectiveness is blunted by enhanced cholesterologenesis within the liver cdl.

Much greater decrements in plasma LDL can be induced by simultaneous administration of an inhibitor of HMG-CoA reductase. This combined therapeutic approach can produce as much as a 50% reduction in plasma choles- terol thereby normalizing the level of this lipid even in severely affected heterozygous familial hype~holesterolaemic patients%.

9 Miettinen, T. (1979) J. Clin. Invest. 64: 1485-1493

10 Vogelberg, K. Ha, Koachinsky, T., Heln, H. and Gries, F. k (1982) Eur. J. Cl&. Phamracol. 22,33-38

11 Kesaniemi, A. and Grundy, S. M. (1984) Arteriosclerosis 4,41-l8

12 Packard, C. J., McKiiey, L., Carr, K. and Shepherd, J. (1983)J. C&n. Invest. 72, 45-51

13 Packard, C. J. acd Shepherd, J. (1982) J. Lipid Res. 23, 1081-1098

14 Lees, A. M., Mok, H. Y. I., Lees, R. S., McCluskey, M. A. and G~ndy, S, M, (1977) Atherosclerosis 28,325-331

15 Schwartzkopff, W. and Jantke, J. H. ~~9~~5~8unck. Med. Wochensch. 120,

16 Heider, J. G. (1986) in Pka~ucaragica~ Conrrol of ffyperlipidaemia (Fears, R., Levy, R. I., Shepherd, J., Packard, C. J. and Miller, N. E., eds), pp. 4%438, Prous SA

17 Be& F. P. and Schaub, R. G. (19861 Arleriosclerasis 6, 42-49

18 Endo, A., Kuroda, M. and Tsujita, Y. (1976) f. Antibiuf. (Takyo) 29,1346-X%48

19 Lee, T-J. (1987) Trends P~~a~aca~. Sci. 8, 442-446

20 Mabuchi, H. et al. (1981) N. Engl. J. Med. 305,478-482

21 Bitheimer, D. W., Grundy, S. M., Brown, M. S. and Goldstein, J.-L. (1983) Prae. Natl Acad. Sci. USA 80,4124-4128

22 Packard, C. J., Clegg, R. J,, Dominiczak, M., Lolimer, A. R. and Shepherd, J. (1986) J. Lipid Res. 27,930-938

23 Shepherd, J. el at. (1984) J, Cfin. &vest. 74,2X4-2177

24 Stewart, J. M., Packard, C. J., Lorimer, A. R., Boas, D. E. andshepherd, J. (1982) Atherasclerasis 44‘355-365

25 Shepherd, J,, Caslake, M. K., Lorimer, A. R., Valiance, B. D. and Packard, C. J, (1985) Arteriosclerosis 5, 162-168

26 Breslow, J. L., Spaulding, D. R., Lux, S. E., Levy, R. I. and Lees, R. S. (1975) N. Engf. J. Med. 293,900-*3

27 Shepherd, J., Packard, C. J., Bicker, S., Lawrie, T. D. V. and Morgan, H.G. (1980) N. Engl. 1. Med. 302,1219-1222

28 ~in~~o~h, 13. R. (1984) Ann. &fern. Med. 101,598-604

Cetaber sodium p-(hexadecylamino)ben- zoate 58.835~ 3-(de~~dinethyIsilyl)-~-~2-(me~hyl- phr.i;*l)-I-phenylthyllpropanamide Octimibate: sodium 8.[(1,4$triphenyl- imidazole-Z-yi)oxy]octanoate