4 Carcinogenicity and Structure in Polycyclic Hydrocarbons

4 Carcinogenicity and Structure in Polycyclic Hydrocarbons D.W. JONES, BSc., Ph.D., F.R.I.C., F.1nst.P.

School of Chemistry, University of Bradford, Bradford, Yorkshire, BD 7 IDP

R.S.MATTHEWS, B.Tech., Ph.D. Department of Chemistry, University of Durham. Durham 1. INTRODUCTION 1.1. Chemical nomenclature of polycyclic hydrocarbons 1.2. Cancer induction and metabolic processes

159 161 162

2. CARCINOGENICITY INDICES 2.1. Methylbenz [alanthracenes 2.2. Other alternant hydrocarbons (AH) 2.3. Non-alternant hydrocarbons

163 163 167 167

3. THEORIES OF HYDROCARBON CARCINOGENESIS 3.1. Mode and conditions of ingestion 3.2. Internal transport 3.3. Initiation mechanism

170 174 174 178

4. APPRAISAL OF INTERCONNECTIONS BETWEEN ELECTRONIC THEORIES 4.1. Electronic energy-level transitions 4.2. Reactivity parameters and structure 4.3. Aromaticity and ring currents: nuclear magnetic resonance spectroscopy

189 189 191 194

5 . CONCLUSIONS

195

IN MEMORIAM

198

ACKNOWLEDGEMENTS

198

REFERENCES

198

1. INTRODUCTION The term ‘cancer’ is applied to a group of multicellular diseases caused by many different agents and, no doubt, involving several biochemical processes. It is 159

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CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

characterized by the growth of malignant cells which proliferate in an apparently uncontrolled way (in contrast to the normal controlled process of cell division), invade adjacent tissues, and disseminate to distant organs to form tumours [ 1, 21. About one-fifth of the deaths in England and Wales are currently ascribed to neoplastic diseases [3], i.e., those associated with the growth of new abnormal body tissues. Of the cancer occurring in man, Boyland [4] has suggested that 90% is caused by chemicals, endogenous or environmental. Chemical (contradistinction physical or viral) carcinogenesis [4a] or cancer induction is the slow process of conversion, initiated by specific chemical carcinogens (cancer-inducing materials), of normal co-ordinated cells into abnormal tissue which can exist independently of the original stimuli. This is essentially a biological, rather than a molecular, definition, Environmental chemical carcinogens may be involved even when tumours in animals (and probably in man) are caused by viruses. Following the observation which was made as long ago as 1775 of an association between cancer and soot contamination of the skin [ 5 ] , polycyclic hydrocarbons were the first compounds to be recognised explicitly as chemical carcinogens. Of the hundreds of diverse organic compounds [6] believed to be capable of inducing cancer in man or in experimental animals, the polycyclic hydrocarbons (together with heterocyclic aromatics), despite their high chemical stability and relative lack of reactivity, are among the most potent and significant classes of chemical carcinogens. As constituents of coal-tars, pitches, tobacco smoke, and pollutants, they are the effective agents in forming human skin cancer [7] ; their importance is underlined by strongly epidemiological evidence of an association [8,9] between cigarette smoking and cancer in man. Carcinogenesis is to be distinguished from inutagenesis (interference with the genetic process). While the two have experimental similarities, they are not necessarily closely related; mutagenic activity can be merely one consequence of the reactivity of a carcinogen or its metabolite. Mutagenesis is a biological phenomenon which is already fairly well-defined in molecular terms [lo] as a perturbation of the genetic code embodied in the sequence of purine-pyrimidine complementary base pairs along the helical deoxyribonucleic-acid (DNA) axis. The aim of this article is to review some of the literature from the past few years on the electronic and structural theories of chemical carcinogenesis by polycyclic aromatic hydrocarbons. The wealth of this literature means that a survey with any pretension to topicality must be selective. There are consequent dangers in a field notable also for spirited controversy (not always free from polemics). Such selection, although partly unconscious, is inevitably influenced by the viewpoint of the authors based on chemical experience, chiefly with solutions, In view of the emphasis on molecular geometry over a wider field of

D.W.JONES and R.S. MATTHEWS

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chemical carcinogenesis in comprehensive reviews [ 11, 121 ,additional attention will be paid here to the situation in solution and to possible links between hydrocarbon carcinogenesis and electronic structure. Indices for the measurement of carcinogenic potency in alternant and non-alternant polycyclics are described in Section 2, followed by a discussion of the chief hypotheses of the mode of carcinogenic action in Section 3. An examination of electronic theories relevant to carcinogenesis of polycyclics in Section 4 precedes a summing-up in Section 5. 1.1. CHEMlCAL NOMENCLATURE OF POLYCYCLIC HYDROCARBONS

The last few years have seen a change in the systematic procedure for designating polycyclic hydrocarbons [13], although the new IUPAC (Rules of 1957) system has been accepted more slowly in the field of this review than in polycyclic chemistry generally. Accordingly, a summary of the IUPAC system will be given to facilitate reference to earlier books and papers involving the Stelzner and Kuh system [ 141,which is, for example, the one adopted by Clar [ 151 . (i) As before, most common parent hydrocarbons continue to have trivial (non-systematic) names [16] ; Rule A-21 of the 1957 IUPAC Rules lists 35 of these. (ii) Structures are drawn with as many rings (except for three-membered rings, all have two sides vertical) as possible in a horizontal line and with the remaining atoms predominantly in the top right quadrant. Substituent positions are then numbered clockwise from number 1, which is the first carbon of the right-hand ring of the top row not engaged in ring fusion. (iii) Where a higher polycyclic hydrocarbon does not have a trivial name, the faces of the largest possible unit with a trivial name [16] are lettereda for face 1-2, and then b, c, etc., successively for all the peripheral faces clockwise. The full name is then constructed by following the name of the substituents, often in abbreviated form (benzo, naphtho, anthra, phenanthro, acenaphtho, and perylo), by square brackets containing the (smallest possible) numbers of the fusion positions (where necessary) in the substituent and the letters of the fusion faces of the larger unit, as in (1). Note that, if necessary, the complete formula is now reorientated to satisfy rule (ii) above and renumbered. It is the replacement of numbers in the old system by letters for the faces of the major parent which constitutes the most obvious change in the nomenclature of hydrocarbons. Formerly, numbers designating faces were listed in front of the substituents, while now the letters designating faces are enclosed in brackets between substituent and trivial-named parent. Examples encountered frequently in the carcinogenesis literature include the changes of what was formerly designated

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

162

i

&$I&

\

‘ /

(4)

1.2-benzanthracene (or sometimes tetraphene) to benz [a] anthracene (1); of

9,10-dimethyl-l,2-benzanthracene (2) (or occasionally 7,12-dimethyltetraphene) to 7,12-dimethylbenz [a] anthracene; of 3.4,5.6-dibenzophenanthrene to dibenzo [c,g]phenanthrene (3); of 1.2,5.6-dibenzanthracene to dibenzo [ah]anthracene (4); of 3.4-benzopyrene (occasionally designated 1.2-benzpyrene) to benzo[a] pyrene ( 5 ) ; and of 1.2-benzpyrene to benzo[e] pyrene (6). 1.2. CANCER INDUCTION AND METABOLIC PROCESSES

The mode of action of polycyclic aromatic carcinogens in initiating epithelial tumours has generally been regarded as ‘direct’ (even though polycyclics are subject to metabolic excretory processes) in that they give rise to tumours in situ [ 171 . For this, solubilization (by proteins, etc.) is a prerequisite, followed by internal transport across the dermis. In the several years which may elapse between exposure to a carcinogen and the emergence of cancer in man, a series of reactions is likely to be involved. In bronchogenic cancer, polycyclic hydrocarbons may act either as proximate agents, forming an active species by metabolic reaction after transport to the active site, or as non-proximate agents, in which formation of the active species precedes arrival at the site of action; again, crossing of the cell wall is involved. Malignancy in neoplastic tissue follows interference with the growth and replication mechanisms of normal cells, but it has not been established which growth agents (proteins, DNA, ribonucleic acid (RNA), histones) are the cellular targets

D.W.JONES and R.S. MATTHEWS

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(receptors) of these chemical carcinogens. Both mutagenic (DNA-carcinogen interaction) and genetic regulation (cytoplasmic protein-carcinogen interaction) hypotheses of initiation mechanism involve the KLM theory of bond reactivity of polycyclic hydrocarbons; all three topics will be discussed in Section 3. When cells are treated so as to increase the activity of the microsomal enzymes which cause metabolic activation, they show increased sensitivity to transformation of hydrocarbons [ 181 and reduced sensitivity to their cytotoxic effect. The observation that hamster cells pretreated with weakly carcinogenic benz[a] anthracene then become more susceptible to the potent carcinogens benz [a] pyrene and 3-methylcholanthrene implies a dependence of carcinogenic activity on metabolic activation. More generally, by physical interaction with tissue components, metabolic activation [19] of a wide range of chemicals, possessing a variety of physical properties and chemical reactivities, yields appreciably more reactive metabolites (products of metabolism). Their combination with tissue eventually leads, it is believed, via general and specific responses, to induction of tumours. Proximate carcinogenic metabolites (which may or may not react with cellular components) have structures which are intermediate between those of the administered precarcinogen and those of the ultimate carcinogenic metabolite which reacts with critical cell constituents and leads to diminished control of cell replication. 2. CARCINOGENICITY INDICES

Several numerical scales of carcinogenicity [20-221 are based on the statistics of cancer induced in small animals, as detected by pathological assay; tumours generally arise preferentially at the site of exposure [23,24]. However, variables are so numerous - animal species, age, strain, sex, hair-cycle, mitotic cycle, diet, as well as the conditions, general toxicity, and site of administration of the test sample - that indices should only be compared when standard procedures have been applied to large numbers of standardised animals (usually specific strains of mice). 2.1. METHYLBENZ[a] ANTHRACENES

For the methylbenz[a] anthracenes (l), careful study by a number of workers has yielded experimental indices which, within the limits of biological testing, are consistent and cover the whole range of carcinogenicity (Table 4.1). Comparison of these indices with those for active dimethylene- and polymethyl-substituted benz[a] anthracenes (Table 4.2) illustrates the effect of substitution

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

164

Table 4.1. COMPARISON OF CARCINOGENICITIES DERIVED BY SEVERAL WORKERS FOR MONOMETHYL-BENZIa] ANTHRACENES

Position of methyl group .___

7

6 8 12 9 10 4

5

1 2 3 11

Percentage sarcoma incidence

Badger indices

a

b

C

93 71 61 52 5

100 100 63 69 0 0 0 0 0 0 0 0

++++ ++++ ++++

5 5 4 3 0 0 0

f

++++

0 0 0 0 0 0 0

d

+++

+ ++ ++ + +

0

+

0 0 0 +?

e

++++

+++

+++ ++ + + + +

+ 0 0 0

a Dunning and Curtiss [25], using rats and subcutaneous injection. Huggins, Pataki and Harvey [ 261, using rats and subcutaneous injection. Haam, E., cited by Dunning and Curtis [25] by Badger index and surface application (epithelial activity). Badger [22), collection of previous results for subcutaneous activity. Subjective average of a-b referred to the Badger scale.

positions and the existence of some approximation to additivity. In the methylbenz [a] anthracenes, fluorine substitution has proved valuable in ascertaining metabolically reactive or carcinogenically important positions [27]. While the activity of methyl derivatives is substantially reduced by substiTable 4.2. CARCINOGENIC INDICES (COLLATED FROM SEVERAL SOURCES) OF ACTIVE POLYSUBSTITUTED BENZ[ajANTHRACENES

Methyl-group positions

Activity

Dimethylene-bridge positions (ace-)

Activity

7.1 2-dimethyl 7,8,12-trimethyl 7,9,12-trirnethyl 7,8,9,12-tetramethyl 8,lZdimethyl 8,9-dimethyl

++++ ++++ ++++ +++ +++ +++

7,8-ace (cholanthrene) 6,7-ace4,s-ace1 1,12-ace

++++ +++ ++

++

D.W. JONES and R.S. MATTHEWS

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Table 4.3. COMPARISON OF CARCINOGENIC ACTIVITY IN DIMETHY L-BENZ [ a ] ANTHRACENES WITH ACTIVITIES OF THE TWO ANALOGOUS MONOMETHYLBENZIa] ANTHRACENES Dimethyl derivative

Activity in dimethyl derivative

Activity in corresponding mono-methyl derivatives (a)

7,12 73 8,12

++++

++++

++++ ++++

++++

+++ +++

++++ +++ +++

6,7 9,lO

+

++++ +

8,11 s,12 3,lO 339 2,10 2.9 1,7

0 0 0 0 0 0 0

7,11 83 6,12

+++ ++

+++

+++ + 0 0 0 0 0

(b)

++

+++ ++ 0

+ ++ +++ + 0

++ +

+ +

+ ++++

tution in the 5-position, substitution in the 3-, 6-, 9-, and 10-positionsis ineffective. This can be held to support either the K-region theory of carcinogenicity (see Section 3.3.1 .) or a ‘lock-and-key’ theory, where an angular benzo-group provides a key. The feeble carcinogenic activity of benz[a] anthracene is enhanced by monomethyl substitution [ l I , 22, 251 in the sequence shown in Table 4.1. Further substitution increases potency (Table 4.2), although 7,8,9,12-tetramethylbenz [a] anthracene is less active than 7,12-dimethylbenz [a] anthracene, the most rapid skin carcinogen. Table 4.3 suggests that dimethylbenz [a] anthracenes display some additivity of the activities of the corresponding monomethyl deriva. tives, except that 1,7-, 5,12-, and 8,11-dimethylbenz[a] anthracenes are surprisingly inactive. For 7- and 12-methylbenz[a] anthracene, methyl substitution at 6, 8 , or 11 has little effect, but substitution at 1, 4, and 5 (i.e. olitside the critical region of 6, 7, 8, and 12 positions) destroys sarcomagenic activity [28]. For 7,12-dimethylbenzanthracene, at least, replacement of all hydrogens by deuterium enhances the carcinogenicity [28a]. Although the metabolites of 12-methylbenz[a]anthracene have been studied (for example, [29]), specific information on the metabolism of the methyl-

i66

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

benz [a] anthracenes in carcinogenesis is not available. The susceptibility of position 7 to an in vivo oxidative process, comparable to that of position 12 in the parent hydrocarbon, suggests that methylation of these positions may reduce the statistical chance of the molecule being eliminated by excretory metabolism. Sims [30] converted 7- and 12-methyl-derivatives into the 8g-dihydroxy and 5,6-dihydroxy compounds by simulated in vivo reactions. The potency of the 6and 8-methyl-compounds, coupled with the weak carcinogenic activity of the 5 and 9-methyl-derivatives, is in apparent support of the ‘protection’ theory of methylation; however, substitution of any one of these sites enhances the carcinogenic activity considerably more than would be expected on ‘protection’ grounds. In this review, carcinogenicities have generally been transformed where necessary (contrast the percentage sarcoma indices of columns a and b in Table 4.1) to the Badger 1221 scheme, in which carcinogenic potency [ l 1,251 is graded as: (i) very marked (+t+t); (ii) marked (ttt); (iii) moderate (++); (iv) slight (+); or (v) nil (0). While errors inherent in biological assays (for example, the use of small numbers of animals and of ill-defined techniques) can contribute to deviations o f t 20% in an index, the above categories are sufficiently broad to be useful. As an illustration both of the leck of corroboration between biological assays and of the variations which occur with changing conditions, Datta and Ghose [31] reported their failure to produce tumours in small numbers of cattle with 2-methylcholanthrene and benz [a] pyrene, which are considered to be notoriously carcinogenic. The same authors noted a similar lack of success with infrahuman primates, while other workers [32] recorded the lack of activity of 20-methylcholanthrene in squirrel monkeys. Iryestation by cattle of small doses of weak carcinogens from virgin or industrially uncontaminated soil [33, 341 can have an anti-carcinogenic effect [35]. Attention is drawn specifically to the small differences (for example, columns c and d in Table 4.2) between testing of carcinogens by epithelial application and subcdaneous injection. Lijinsky, Garcia and Saffiotti [36] found that partially hydrogenated derivatives of several polynuclear hydrocarbons showed different tumorigenic activity from tests by the two methods. The increased mortality rate of subcutaneously injected mice is probably a consequence of other toxic effects by these hydrocarbons. On the face of it, the apparent lack of tumour specificity displayed by polycyclic hydrocarbon carcinogens means that there is no great need to differentiate between types of tumour generated at different application locations. It is possible, however, that hydrocarbons differ according to the distributions of tumour types to which they give rise.

D.W. JONES and R.S. MATTHEWS

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2.2. OTHER ALTERNANT HYDROCARBONS (AH)

Several reviewers have partially collated the carcinogenic indices of aromatic hydrocarbons and their derivatives; the activity of the monomethylbenz [ a] anthracenes has been discussed in the previous section. Badger [22] reviewed the results of screening for carcinogenicity up t o 1948; Table 4.1 shows that these are generally in accord with more recent data. Hartwell [37] and Shubik and Hartwell [38] surveyed the results of tested compounds comprehensively. While there have been few collations of the results of toxicological carcinogenic screening in the last decade, there are many reports on the activity of individual hydrocarbons [39] or specific derivatives [40] , particularly as a result of tobacco-smoke research. Of the six purely-aromatic tetracyclic hydrocarbons, only chrysene, benz [a] anthracene and benzo [ b ]phenanthrene possess even marginal activity; but, as shown (Tables 4.1,4.2, and 4.3), many benz[a] anthracene derivatives are active, as are some benzo[c] phenanthrene derivatives. Substitution of one, two, or three methyl groups at 6, 7, 8, or 12 in benz [a] anthracene markedly increases activity [41]. Studies of N-mustard derivatives of anthracenes and benz[a] anthracenes [42] emphasise the parallelism between carcinogenic and carcinostatic properties of poly-nuclear derivatives [43]. The marked influence of a small change in chemical structure is illustrated by the sequence of anti-tumour activities 10-ethyl-9-anthryl > 9-anthryl >> 10-methyl-9-anthryl for the change of nzesu substituent from Et t o H t o Me [42]. Of the fifteen pentacyclic hydrocarbons, five are known sarcomatogens and two are borderline cases; again, several derivatives are active. Biological screening results are less complete for the hexacyclic aromatic hydrocarbons. Only the well-known carcinogens are listed in Tables 4.4 and 4.5. Table 4.4 lists the active alternant, parent polycyclic aromatic hydrocarbons under the names currently accepted; Table 4.5 gives the activities of tested methylene derivatives which possess structures similar t o those in the homologous series of alternant parents. While the time is appropriate for a new compilation of carcinogenicity data and a reassessment of the activity of polycyclic hydrocarbons and their derivatives, this will not be attempted here. 2.3. NON-ALTERNANT HYDROCARBONS

Screening of non-alternants (NAH’s) is much less extensive than for alternants; tested parents are listed in Table 4.6. Knowledge of the carcinogenesis of NAH’s could be valuable since, unlike molecules containing heteroatoms, NAH’s are likely t o have a similar carcinogenic mechanism t o that of AH’S; consequently, only physico-chemical correlations which can readily include NAH’s are accept-

168

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS Table 4.4. CARCINOGENICALLY-ACTIVE PARENT, ALTERNANT AROMATIC HYDROCARBONS * Structure

Nomenclature

Activity

Tetracycl i c aromatic hydrocarbons

Benz[a]anthracene

O/+

Benzo[s]phenanthrene

t

Chrysene

Pentacyclic aromatic hydrocarbons Benz [ a l p y r e n e

(12)

+tt+

Dibenzo[a-,~]anthracene

+t

Eenzo[a]chrysene

t/tt

Benzo[f]chrysene

t

Dibenz[a,c]anthracene

o/t

169

D.W. JONES and R.S. MATTHEWS Table 4.4 (continued)

Structure

Nomenclature

A ctivitv

H e x a c y c l i c a r o m a t i c hydrocarbons

(17)

D i benzo [c,l]PYrene

tt+

D i b e n z o [g,h]PYrene

+t+

D i benzo [g,g]pyrene

++++

(20)

D i benzo [g,L]pyrene

+++

(21)

Dibenzo [g,c]tetracene

o/+

H e p t a c y c l i c a r o m a t i c hydrocarbons

A

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CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

Structure

Nomenclature

Activity

Tri benzo [c,g,l]pyrene

tt

Tri benzo[ 5,5,J] tetracene

o/+

-~ ~

1. References [ 11, 22, 36-38 and 44471 2. The diagramatic representation of a circle inside a hexagon indicates arornaricify and not necessarily a sextet of electrons in that ring.

able. Furthermore, methyl AH’s and analogues containing cyclopentadienyl and indene rings may have similar mechanisms. Heiger found that the basic tissue constituent cholesterol, a hydrocarbon with one polar group, is weakly but surprisingly tumorigenic 1471 ; recently, cholesterol has been shown to form an equimolar complex with lecithin when dispersed in an excess of water [48] . Comparison of AH and NAH carcinogens suggests that the benzo-group is an essential feature. Substitution at the adjacent position, or similar positions, leads to large potency changes, as in the 5, 7, 12 positions already referred to in the benz [a] anthracenes. If the benzo-group is intercalating in a biomolecule, the lipophilic character of the uninvolved residue would be of paramount importance; the ‘lock and key’ terminology suits this pictorial description. The consequent proximity of cellular and aromatic molecules may lead to K-region bonding (see Section 3.3.1). In Section 3, some of the mechanisms postulated for carcinogenicity are discussed with particular reference to these AH’s and NAH’s. 3. THEORIES OF HYDROCARBON CARCINOGENESIS

Much of the research on the mechanism of chemical carcinogens is inspired by the hope of controlling, or even reversing, malignancy (interference, which is at

D.W. JONES and R.S. MATTHEWS

171

Table 4.5. TESTED METHYLENE DERIVATIVES OF ALTERNANT HYDROCARBONS

w ?&c q

Structure

Nomenclature F1 uorene

Activity

0

Benzo[c] f l uorene

Benzo [a]f 1uo r e ne

Dibenzo[a,g]fluorene

+++ ( ? )

Dibenzo[c,p]fluorene

o

Dibenzo[a,c]fluorene

o/t

Dibenzo(a,L]fluorene

O/+

Cholanthrene

t

5,6-ethanochol anthrene (7,8; 11,12-diethanobe nz a n t h r a ce ne )

+++

[a]

172

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS Table 4.5 (continued)

Structure

Nomenclature

Activity

1,2,3,4-tetrahydrodibenz[a-,t~]anthracene

+++

(35

5,6-dihydrod i benz [g,h]anthracene

t

(36)

dibenz[a,t~]anthracene

1,2,3,4,12,13-hexahydro-

+

(37)

5,6-di hydrodibenz[a,.j]anthracene

+tt

1,2,3,4-tetrahydrodibenz[g,.j]anthracene

o/+

1,2,3,4,8,9-hexa hydro d i benz [c,,j]anthracene

0

For references see Table 4.4

least quasi-permanent, with the growth and replication mechanisms of normal cells) in neoplastic tissue [35, 491. It has tended to be directed at the chemical reactivities of the carcinogens and their metabolites [50] so as to determine first the active forms or ultimate carcinogens and secondly the nature of their interactions with tissue. The question of which (if any specific) growth agents are involved as cellular targets of carcinogens, has not yet been resolved, despite the efforts of several groups [lo, 51, 521 . Some of the factors on which the carcino-

D.W. JONES and R.S. MATTHEWS

173

Table 4.6. TESTED NON-ALTERNANT PARENT HYDROCARBONS ~

Structure

&8

(47)

~~~~

Nomenclature

~~~

Activity

F1 u o r a n t h r e n e

0

Benzo[kJfluoranthrene

O/+

Benzo[.j]fluoranthrene

+++

Benzo[b]fluoranthrene

+++

Benzo[g,h,L]fl

uoranthrene

0

Phenylene[P,!]

pyrene

+

Dibenzo[a,g]fluoranthrene

+

Dibenzo[b,k]fluoranthrene

0

174

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

genic index of a polycyclic hydrocarbon depends will be discussed in turn. While the theories associated with each factor will be treated in isolation, it should be emphasised that they may be interdependent. 3.1. MODE AND CONDITIONS OF INGESTION

The influence of external solvent on experimentally-obtained indices is not always recognised. From experiments with croton oil (a fixed seed oil) as solvent, Berenblum [53] suggested that carcinogenesis occurred in two stages, initiation and promotion; solvent could also affect activity by penetration of cell or cellular organelle (part of a cell with specialised biological function) to varying degrees. While stimulation of DNA-RNA activity by croton oil has been confirmed [54], no link with tumour initiation has been established. Previously, it had been assumed that a carcinogen initiated carcinogenesis and that croton oil promoted or encouraged tumour formation by a sensitising effect, i.e., co-carcinogenesis as opposed to anticarcinogenesis. Phorbol and its derivatives have been identified as the active constituents in croton oil [55, 561. The inactivity of 20-methylcholanthrene in lanolin [57], coupled with its activity in benzene, lard, and sebum (fatty secretion of the sebaceous glands) may be a consequence of the extent to which solution or micellar formation aids or inhibits cell/organelle penetration. By indicating differences between interactions in solvent systems similar to those mentioned above, proton magnetic resonance spectroscopy (see Section 4.3) can facilitate recognition of the significant interactions, whether non-specific (hydrophilic or hydrophobic) or specific. 3.2. INTERNAL TRANSPORT

Aside from whether they are directly active per se, or merely as precursors of active metabolic intermediates, carcinogenic polycyclic hydrocarbons can initiate carcinomas (e.g. epithelioma) at the point of application on the skin (the commonest mode of study), or can lead to distant tumours, as in bronchogenic cancer [23] . The mechanism probably includes solubilisation of the carcinogens (by proteins, nucleoproteins, fatty acid esters, etc. or even in aqueous solution [SS]), so as to enable them to traverse the dermis (possibly via an external solvent) and thus react in the epidermis, which possesses a lipoid barrier. Alternatively, appendageal transport routes (hair follicles, sweat glands, sebaceous glands) may be used [59]. When carcinogens act at a distance, as with liver, breast, bladder and other internal cancers, an internal transport mechanism or medium is evidently needed. Whether the carcinogen is acting directly on the target tissue or indirectly, the

D.W. JONES and R.S. MATTHEWS

175

crossing of the cell membrane or wall, or lipoid barrier in the case of skin [60], can be accomplished by direct intrusion (in a vacuole) as a complex with an essential cell component which has a natural cell-intrusion mechanism. This little-studied physiological process termed endocytosis (or invagination at an active site) [61, 621 is a problem of transport kinetics [ 6 0 ] .Electron and optical microscopy reveal endocytotic vesicles (or vacuoles) in a wide range of sizes. Particles can also cross cell walls by direct penetration of membrane structure [63] ; for ions, penetration depends o n their size but, for hydrocarbon molecules, penetration of the lipoprotein matrix depends on partition co-efficients [ 6 3 , 641, as is supported by studies with representative polar-non-polar solvent systems [65]. If sufficient quantities o f lipoprotein matrix could be separated and dispersed in an aqueous medium, intermolecular interactions [66] might be studied by proton magnetic resonance spectroscopy. Cell walls, or the outermost layers of the cytoplasm of a cell, are composed of orientated phospholipid and protein molecules called lipoproteins [67, 681 , while the ‘essential cell component’ mentioned above in connection with hydrocarbon complexing (the carrier) is probably also a protein. Aqueous-organic phase partitioning must be relevant since a carcinogen may be partitioned several times between aqueous and organic phases during its passage through tissue; the lipo-hydrophilic character of polycyclic hydrocarbons has been investigated, therefore, as has its correlation with biological activity [65, 691. Hansch and Fujita [65] also correlated lipo-hydrophilic character in 47 polycyclic hydrocarbons with carcinogenicity: large molecules are too lipophilic (or not soluble enough) and small molecules insufficiently lipophilic for activity. The influences of position and type of substitution o n lipophilic character are also in general agreement with observed carcinogenicity. If it is accepted that both transport and cell-membrane (lipoid-barrier) crossing by carcinogenic hydrocarbons are necessary, these lipophilicity correlations provide a facile explanation for relative carcinogenicities. Such an explanation has the merits of retaining the same interaction mechanism (except, sometimes, t o account for extremely soluble or insoluble cases) and of consistency with theories in which hydrocarbons have general cell-disrupting activities, rather than commonly-accepted specific interactions. Conversely, within the limits of partition-dominated molecular concentrations, some drugs are beneficial because of enhanced hydrophilic character (aqueous media solubility), for example, hydrophilic nitrobenzamides are carcinostatic [70] . Since Demisch and Wright [71] measured partition coefficients with respect t o a solution containing monoethanolammonium desoxycholate (rather than a purely aqueous phase), their linear carcinogenicity relation is not strictly comparable with the parabolic one obtained by Leo, Hansch and Elkins [64] (see also Section 4.2).

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CARCINOGENICITY IN P0LYCYCI.IC HYDROCARBONS

The suggestion, following calculations from hydrocarbon models (rather than diffraction measurements, of which there is a paucity), of a link between carcinogenicity [26, 721 and the non-planarity of substituted benz [a] anthracenes, is probably a fortuitous consequence of the small number of compounds investigated. Deviation from planarity, as with 7,12-dimethylbenz [a] anthracene and benzo [c] phenanthrene has been found to curtail or eliminate enzyme-inducing activity [73]. Planarity is likely to be related to the solubilisation-transport problem. Franke [74] has revived suggestions that hydrocarbons bind to proteins by reducing hydrophobic aromatic-hydrocarbon areas and covering hydrophobic protein surface; activity is lowered when bulky substituents reduce hydrophobic contact areas in size and strength. Earlier, he calculated [75] an index, K , , for the enthalpy of binding of polycyclic aromatic hydrocarbons to human serum albumin (protein) both directly and indirectly from the free energy of solution in water and from molar refractivity. This approach seems to be especially valuable in its attempt, by regression analysis between carcinogenicity and both K , and chemical reactivity, to rationalize two parameters; correlations between several parameters are generally more realistic than monoparametric treatments. Franke calculated acceptable multiple correlation coefficients (0.93) for 34 hydrocarbons. For the monomethylbenz [a] anthracenes, however, log K , values alone (rather than log K , + log K ) extracted from Franke’s measurements give no correlation with carcinogenicity, even when HMO reactivity indices for the K-region are taken into account [76] . There is an essential difference between the transport theory of Hansch and Fujita and that of Franke. Hansch and Fujita regard the kinetic balance of solubilities in different phases as paramount; substitution in these hydrocarbons to increase the hydrophilic character is generally accompanied by a reduction in carcinogenic activity [ l 11 ; absolute solubilities of the compounds are not involved. Franke, on the other hand, considers only the enthalpy of solutions in the lipid phase (equivalent to protein binding). No doubt, a more comprehensive theory will combine these approaches with terms not only for the absolute solubilities in aqueous and lipid phases but also for their relative magnitudes. Terms from (a) hydrophilicity (or from hydrophobicity) and from (b) lipophilicity (or lipophobicity) would be additive constitutive properties, referenced with respect to some standard compound. In this way, both solubility and the hydrophobic binding character of Franke might both be incorporated while Hansch’s treatment of partition functions could be expressed in terms of (a) and (b). Recently, Leo, Hansch and Elkins [64] have reviewed ways of determining such functions and thermodynamic changes when there was a major structural alteration in the parent molecule. Among other physical studies, Fuson and Josien (771 report the infrared spectra of the methylbeflz[a] anthracene series in

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nonpolar solution; an aromatic stretching vibration ca. 3050 cm- (presumably the 5,6-bond, although the assignment is not explicit) shows some correlation with carcinogenic activity. [The fluorescence, ultraviolet and visible spectra of the series were reported in Russian [78] ; spectral shifts are related directly to LCAO MO calculations. Following the correlations between carcinogenicity and theoretical molecular electronic transition energies made by Mason [79-8 1] , Birks [82] , and Steele, Cusachs and McClynn [83, 841 and in part experimentally confirmed by Sung and Lazar [85], the Russian results emphasise the importance of electronic transitions in the carcinogenic involvement of the series.] Introduction of polar substituents into aromatic hydrocarbons will influence hydrophobic-lipophilic forces when electron donating-withdrawing effects of polar and bulky substituents may be important [ l 11 . In particular, Arcos and Arcos [ 11] noted that increasing hydrophilicity of polycyclic hydrocarbons will attenuate carcinogenic activity. In 7,12-dimethylbenz[a] anthracene (2), which is active, the methyl groups may radically enhance the hydrophobic character, by comparison with the inactive parent, and lead to (undefined) intermolecular forces favouring solubilisation by proteins. A similar but unfavourable binding effect is expected in large hydrocarbons, with a consequent reduction in aqueous solubilisation, as partition coefficients confirm [65, 741. In the absence of details as to the proteins involved, such conclusions must be very tentative. Further, the few large hydrocarbons which are carcinogenic [86, 871 would require very large protein binding areas (the hydrocarbon encumbrance areas of Arcos and Arcos [ 11 ] ). Hydrocarbon-protein binding is often plausibly thought [ l 11 to depend on the shape and dimensions of the protein-surface receiver site, in accord with fortuitous shape similarities of hydrocarbons and steroids [88] ; this does not invalidate the hydrophobic-bonding theory. However, carcinogenic polycyclic hydrocarbons embrace a wide range of shapes and sizes (from three [89] to seven [90] fused rings). Carcinogen-protein interaction could be based on the inclusion of polycyclic carcinogens between hydrogen-bonded layers of polypeptide chains as are found in DNA. Harvey and Halonen [91] noted that the strength of binding of carcinogenic hydrocarbons to nucleosides is proportional to the number of fused aromatic rings. Sung and Lazar [85] provided confirmation of Mason’s [79, 801 controversial hypothesis of hydrocarbon binding to proteins, which is based on the the so-called Brillouin-Bloch bands in proteins (primarily the delocalised bond in peptide moieties); the energy difference between the highest occupied electron band and the first unoccupied (so-called conduction) band is approximately 3 eV. Mason observed that, for carcinogenicity in polycyclic aromatic hydrocar-

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CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

bons, the theoretical (Hiickel-molecular-orbital) energy-level differences between the highest occupied orbital and the second unoccupied orbital must be 3.24 k 0.1 1 eV. Several consequent disruption theories have been modified and criticised [92]. From a similar correlation between carcinogenicity and the mean energy of the first excited singlet state, where E = 3.04-3.20 eV for carcinogenicity, dipole-dipole transfer of %excitation energy has been suggested [82] . Sensitivity of the semi-conductivity of bovine plasma albumin gels under high vacuum to small concentrations of carcinogens provides substantial evidence for electronic interactions between proteins and aromatic hydrocarbons. Although correlations with carcinogenesis are lacking, electronic perturbations of protein systems may well be involved in carcinogenesis, whether directly or in connection with transport-complexing processes [93] . These correlations [82] are discussed further in Section 4 because they have much in common with reactivity theories; indeed, uncertainty about the basic causative interaction (theories of which are reviewed in Section 3.3) often causes the boundaries between transport and reactivity theories to be blurred. Interestingly, studies of organic charge-transfer complexes, for example of methylbenz[a] anthracenes with 2,4,7-trinitrotoluene [94] , which involve partial exchanges of an electron from donor (protein or nucleic acid) to acceptor, have revealed little evidence of any direct relation [95] (i.e., on a monoparametric basis) between charge-transfer complexing [94, 96, 971 and the controlling processes of carcinogenesis or indeed of other in vivo processes [98, 991. Any such influence of complexing would be via the solubilization of hydrocarbons by DNA, DNA bases, and other proteins which has been widely investigated [ 100- 1021 . Giovanella, McKinney and Heidelberg [ 1001 undermined support for earlier suggestions [ 1031 of direct non-enzymic interaction of benz[a] pyrene with DNA in vitro by pointing out that Boyland and Green’s solubilization [lo31 was actually the formation of an aqueous colloidal suspension of hydrocarbon stabilized by DNA. Absence of experimental observation does not necessarily contradict the association of free radicals and/or complexes with carcinogenesis [104], whether in the transport or (as discussed in the next section) in the initiation process. 3.3. INITIATION MECHANISM

Whether complexing or reaction of the carcinogen in the cell is responsible for tumorigenesis, the nature of the cellular target protein, nucleoprotein, desoxyribonucleic acid (DNA), ribonucleic acid (RNA), histone, or other cellular moiety - is unknown. While previous reviews have covered investigations into cellular mechanisms in general [ l l , 22, 35, 44, 105, 1061, the present review is confined to polycyclic aromatic hydrocarbons. ~~

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Two basic kinds of mechanistic hypothesis to account for changes in cellular behaviour concern, respectively, DNA-carcinogen interactions (mutagenic theories, whereby the information contained in the DNA molecule is altered) and protein-carcinogen interactions (genetic-regulation theories, whereby specific proteins or RNA’s are altered to produce heritable changes in genome expression [50]). In addition, the immunological and latent virus theories are not specifically associated with polycyclic hydrocarbons. Any discussion of the mechanisms at cellular and molecular level must include a significant theory of bond reactivity which can be correlated with carcinogenicity, the K and L theory.

3.3.1. Theory ojthe K andL region The tendency of most potent carcinogenic hydrocarbons to undergo addition reactions, as well as oxidations and reductions, at sites analogous to the phenanthrene 9,lO-bond (48) caused these reactive ‘K regions’ to be designated as reaction centres [107]. This led to the first major application of molecular orbital (MO) theory to biological structure-activity relations [ 1081. Indeed, whether or not polycyclics differ among themselves in the sequence of metabolic activation and subsequent carcinogenic action, they remain the group of carcinogens most amenable to MO theory as a probe of relations between structure and activity. In the absence of complementary studies with other variables such as solubility, correlations between reactivity and carcinogenic activity [22, 1091 are usually limited. 2

L

Cellular receptor

Developed from threshold electron-density concepts [l 10, 1I I ] , the K- and L-region theory was extended [45, 1121 with the help of localisation theory [ l 131 . It relates carcinogenicity indices with three features (49) of electronic structure of carcinogens [27,44, 1061 : (a) olefinic K region, for example, the 5,6-bond of benz[a] anthracene; (b) transannular L region, for example, the 7,12 region of benz[a] anthracene; and

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(c) M region, for example, the 3,4 region of benz [a] anthracene; of these, the first two are the most significant. For carcinogenic activity, the K region must be reactive, i.e. the ortho-localisation energy must be smaller than 3.3 1 (threshold value), where p is the Hiickel resonance integral; and the L region (from atoms with large atom localization energy) must be unreactive, i.e. para-localisation energy greater than 5.66 p. These limiting values can account for the activity of many benzoid hydrocarbons. Presumably some type of cellular complex initiates carcinogenesis and the high-electron-density K region donates electrons (50) to a cellular receptor, leading to covalent bonding between the different entities. Flurry’s [ 114) simple evaluation of ortho- and para-localisation energies is applicable only to unsubstituted, alternant aromatic hydrocarbons. Hoffman [ 1 151 applied more advanced self-consistent-field (Pople-Pariser-Parr) molecularorbital (SCFMO) calculations of chemical reactivity and carcinogenicity to 41 polycyclic hydrocarbons.

(51)

0.0

Two complex indices of reactivity, (i) ortholocalisation energy (OLE), for the K region, and (ii) paralocalisation (PLE) energy, for the L region, are defined as (i) bond localisation energy (BLE) plus the cation-localisation energy (CLE-minimum, or lowest of the two-atom localization energies); and (ii) paralocalisation-of-electrons energy (PLEE) plus cation-localisation energy (CLE-minimum).

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The first term is important and the results are expressed in terms of the resonance integral, 0,via a Linear Combination of Atomic Orbitals (LCAO) method. These indices are a measure of the change in n-energy of a transition state in an electrophilic reaction where the electrons become located (51); the transition systems are calculated with two electrons less and one or two atoms less than the parent. Equivalent predictions of the ranking of hydrocarbon carcinogenicities were made by Mainster and Memory [ 1 161 via superdelocalisabilities (S,)as measures of reactivity. For activity, the sum of S, in the K region, must be ,Z 2 2.05, and the L region sum, I , < 2.30. This complements similar studies, based on S, values, by Nagata [117] and Kouteckf and Zahradnik [118] . Suggestions that hydrocarbons are carcinogenic by virtue of their ability to exist in radical form were made in 1942 by Kensler, Dexter and Rhoads [119]. Interest in this was revived by the observation [120, 1211 of electron spin

resonance (e.s.r.) signals in tobacco-smoke tars; about one-sixth of the observed radicals were stable. Soot, a known carcinogen, contains free radicals [120], and benz[a] pyrene yields free radicals when heated. More directly, there is evidence [122] for the possible existence of free radicals in vivo after excised rabbit lung has been exposed to cigarette smoke. Free radicals are generated in many pyrolysed or charred food-stuffs, although their effect in vivo (in the digestive tract) is unknown [ 1231 . Radical cations of aromatic carcinogens, prepared and trapped both from oxidation [ 1241 and from complexes with tetrachloro-o-benzoquinone [ 125, 1261 ,led to the suggestion that free radicals from complexes in vivo are carcinogenic initiators. Evidence of free radicals in such systems is well-documented although not

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in in vivo tests. For instance, n o e.s.r. signals were detected [127, 1281 from carcinogen-DNA or RNA complexes, and n o correlation was found between the e.s.r. signals and carcinogenicity of N-carcinogens and tetracyanoethylene [ 1291 . Griffith and Poole [ 1301 admitted the difficulties of correlating e.s.r. hyperfine splitting constants of aromatic cations with carcinogenicity. The carbonium ion resulting from M-region oxidation [ 13 1] degrades to enolic products, as shown in 52-55, easily excreted from the cell, whereas the more stable K-region carbonium ion is less easily eliminated and can react, with essential cell constituents t o give covalently-bound substituents. Thus the structure and stability of the metabolically-formed reactive intermediates, and the consequent ease of their elimination from the cell, can influence the observed carcinogenicity. As shown earlier [27], the 5-position in benz[a] anthracene is a reactive focus for carcinogenic action [131] ; while fluorine substitution has no effect on the 6-position, in the 5-position it markedly reduces activity. Poole and Griffith's [ I321 reactivity table also indicates that position-5 has a greater spin density (HMO) than position-6 and is more susceptible t o electrophilic reagents. In a series of methyl derivatives of odd-alternant hydrocarbons [ 13 1 ] , ionic stability is roughly correlated both with the coefficient (a",) of the non-bonding MO at the site of carbonium-ion formation and with carcinogenic potency. This correlation gives similar predictions t o those of Pullman; conversely, Pullman's indices implicitly define the stability of the carbonium-ion intermediates. While Sims [17] considered that analogous intermediates (56a) based o n the formation of a K-region epoxide species [ 1331 during metabolism were potential carcinogens, Dipple, Lawley and Brookes [131] regarded then as less important than the carbonium ions. These viewpoints may not be inconsistent if the three structures 56a, b , and c are rapidly interchanging or equivalent forms of the

same species. Knowledge of epoxide species as intermediates in the metabolism of polycyclics [134, 1351 has recently been advanced in two ways. Although oxidised hydrocarbons had been observed in metabolic reactions before, there is now evidence for the production of epoxides of methylcholanthrene, phenanthrene 11361, benz[a] anthracene [137] and dibenz[a,h] anthracene [138] as intermediates in the metabolism of cell microsomes. Secondly, these epoxides of

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methylcholanthrene and dibenz[a,h] anthracene [ 1391 are mutagenic (and also cytotoxic) in proportion to their carcinogenicities. This points towards intracellular metabolism as activating ostensibly unreactive polycyclic hydrocarbons [138]. For interactions of oxygen molecules with the K-region, Steele, Cusachs and McGlynn [83] find that carcinogenic activity reaches a maximum when the lowest excited singlet molecular-energy-level term (which is as low as 0.98 eV) and the lowest triplet level in a polycyclic hydrocarbon differ by 1 eV. If transient complexing occurs between aromatic carcinogens and oxygen molecules, charge transfer may initiate or aid intermediate formation. By ultraviolet absorption spectra, Gemant [ 141] measured the sequence of oxidisability by inorganic compounds of such carcinogenic polynuclear hydrocarbons as benzo [a] pyrene, 7,12-dimethylbenz [a] anthracene, 20-methylcholanthrene, benz [a] anthracene and dibenz [a,h]anthracene. These indicated that oxidation might be a first step in carcinogenesis, followed, in view of enhanced complex stability after light-

20

@@ 00

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CARCINOGENlCITY IN POLYCYCLIC HY DKOCARBONS

oxidation, by protein complexing o f the hydrocarbon. The hypothesis of transferable excitation energy from chemiluminescent reactions for inducing interaction between carcinogen and cell substrate, proposed for biological reactions, could perhaps also be applied t o the above oxidation reactions [141a] . Such. in situ energy exchanges may be defined as ‘dark’ photochemical reactions [ 141b] . The biochemical significance of the K- and L-regions is that carcinogenesis involves a K-region interaction (whether complexing or chemical reaction) with a cellular receptor, while the L-region detracts from carcinogenic activity, via metabolic elimination. Exceptions to the K- and L-region theory include the activity of 1,2-diethyldiphenylethene [ 1091 . Although anthanthrene (dibenzo [d,e,j)~,n,o] chrysene possesses active K regions (Pullman calculation), it is carcinogenically inactive; the pair of active methine regions may behave as a pseudo-L-region (57), methylation of which yields an active derivative [ 1421 . Other polycyclic aromatic carcinogens without K-regions [ 1 I ] include mesodihydro-cholanthrene (++) and nieso-dihydro-20-methylcholanthrene (++) (58), and also 3 &dihydrodibenz [a.h]pyrene (59). The heptacyclic tribenzo [a,e,i] pyrene (23, Table 4.4) [143], with both K-region protons in bay positions, is a surprisingly potent carcinogen, especiakly in view of its relative insolubility, even in the most versatile organic solvents. Further, dibenz [a,c]anthracene (14) (Table 4.4), previously thought inactive, with an active L region but no K region, is now known [36] t o be weakly active, as is 1O-methylbenz[a,c]anthracene [144]. Additionally, none of the carcinogens 11,12-dihydro-3-methyl1,2,3,4,12,13-hexahydrodibenz[a,h] anthracene 1361 , or cholanthrene, 9,lO-dimethylanthracene [36] possesses a K-region. Many heterocyclic analogues of the aromatic hydrocarbons, e.g. 60-63 [145], do not possess formal K-regions and yet are potent carcinogens.

3.3.2. Protein-elimination or genetic-regulation theories As indicated in Section 1.2, much evidence has been accumulated in apparent support of, respectively, the mutagenic or DNA-carcinogen interaction (Section 3.3.3) and genetic-regulation or protein-carcinogen interaction theories of carcinogenesis initiation by polycyclic hydrocarbons; both approaches utilize the KLM theory of bond reactivity of polycyclic hydrocarbons (Section 3.3.1). In principle, the two types of interaction by metabolites are presumably not mutually exclusive. Throughout the 1950’s, protein-carcinogen interaction, discussed in this section, was regarded by many workers as fundamental t o carcinogenesis. It would now seem likely that the covalent reaction of a carcinogen with nucleic acids and proteins needs either chemical activation 11461 or prior metabolism by a microsomal system 11471.

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Genetic-regulation theories depend on evidence [ 1061 for in vivo proteincarcinogen complexing; this is generally much stronger than the binding of carcinogen to DNA (Section 3.3.3). Carcinogenicity has often been correlated with ability to complex proteins [148, 1491, although Brookes and Lawley [ l o l l and others found no experimental correlation between carcinogenic potency and binding to cellular proteins. Binding of carcinogens (or their metabolites) can be studied by spectrophotometric and radiochemical methods; for in vivo studies, starch-gel electrophoretic techniques [50, 5 11 are advantageous in that carcinogens and non-carcinogens bind to different electrophoretic fractions of proteins. Dibenz [a,h] anthracene (or 1.2,3.4-dibenzanthracene) is an example of a non-carcinogen binding strongly to protein. In one important fraction, termed Fraction I [ 1061 or Fraction 12 [ 1051, binding is proportional to hydrocarbon activity; in another, activity or non-activity appeared the only criterion for binding or non-binding. Fractions giving indiscriminate binding may be associated with detoxification and other metabolic functions [151]. Absence of the complex or protein fraction in tumows [52] favours a protein-deletion theory of carcinogenesis (see below). Sorof, Young, McCue and Fetherman [52], who also separated proteins by column electrophoresis, found that bound dyes and 2-acetamidofluorene were localised in distinct fractions; these workers’ L protein has nearly the same electrophoretic mobility as that of the soluble proteins of mouse skin to which carcinogenic hydrocarbons bind specifically. More recently, the sub-units were separated and their molecular weights were determined. Involvement of the K-region of binding appeared to be shown by dissociating protein complexes of radioactively-labelled dibenzanthracenes [ 152,1531 (64); 30%of the radioactivity was recovered as noncarcinogenic [ 1491 2-phenylphenanthrene-3,2’-dicarboxylic acid. This may not be true in the light of later work [ 1541 ; even if it is, this may be merely a consequence of detoxification and does not necessarily imply that K-region blocking reduces activity.

If covalent binding of polycyclic hydrocarbons to soluble proteins (missing from tumours) in target tissues occurs in proportion to carcinogenic activity

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CARCINOGENICITY I N POLYCYCLIC HYDROCARBONS

[105], a cancer-inducing mechanism is inferred. Much of the evidence on protein-carcinogen binding appears to support the protein-deletion theory of carcinogenesis induction. According to this, chemical binding of carcinogenic compounds to tissue proteins causes the cells generated by division to be deficient in these proteins [105, 106, 1511, assumed to be growth-control enzymes, so that neoplastic tissue can develop. In response to criticism [lo51 , this simple explanation of cytoplasmic deletion leading to permanent change has been modified to incorporate modern biological concepts. The stringent (and mirror-image) geometrical requirements on hydrocarbons for repression of an enzyme synthesis and for induction of microsomal oxidases have been emphasized and regulation of microsomal oxidase levels may be by cascade-coupled operon systems [73]. There is some support [I 551 for the perpetuated-change hypothesis [155, 1561 ; Figure 4.1 gives the basic regulatory system proposed for enzyme induction and repression. Although it was suggested to explain cytoplasmic inheritance, Heidelberger’s [ 1051 basic product-perpetuation circuit does not account for the observation in tumour experiments that transient action of a carcinogen

( A ) Inductive c i r c u i t I

7 Operon

Operator gene

S t r u c t u r a l gene

/

-I

I

t

0 Exogenous r e g u l a t o r molecule,

E

i n i t i a t i n g enzyme induction

m-RNA

Enzyme

( B ) Repression c i r c u i t

I

Regulator gene

I

l

f

a

I

m-RNA

Endogenous

~

Operator gene

1

aporepressor

0 Exogenous r e g u l a t o r molecule, repressing enzyme production

Figure 4.1.

S t r u c t u r a l gene

&rn-RNA t E

Enzyme

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D.W. JONES and R.S. MATTHEWS

can give malignant growth, i.e. inheritance. Pitot and Heidelberger [I551 therefore devised and modified a regulated product-perpetuation circuit (Figure 4.2). In the absence of carcinogen, the circuit for production of enzyme E is permanently off; the aporepressor activates operator gene I, producing a repressor which shuts down operon 11. Introduction of a carcinogen inactivates repressor Operon I

Operon II

Regulator gene m-RNA

3 Aporepressor

i-

Regulator molecule

I

I

I

I

I

Exogenous carcinogen 0 -

I Substrate

Figure 4.2. Modified product perpetuation theory (Pitot-Heidelberger)

X and causes operon I to open; consequently, a regulator molecule (0) is produced (from enzyme and substrate), which inactivates the aporepressor and stops repressor production from RG2. Thus operon I is always open after transient carcinogenic action. Several analogous proposals [1051 depend on epigenetic phenomena. Results from theoretical study with analogue [157] and, particularly, digital computation [ 1581 were consistent with irreversible operon blocking. Theoretical models have recently been considered [1591 whereby polycyclic hydrocarbons control induction and repression of microsomal drugmetabolizing enzymes by transcription or translation. 3.3.3.Mutagenic theories The stability of malignant transformation induced by chemical carcinogens suggests that chemical carcinogenesis results from chemically induced mutation [ 1601 . Less is known about mutagenic than about genetic-regulation theories; interaction with genetic-code material (DNA), rather than with other inter- or intra-cellular materials, is involved. Until recently, at least, conventional assay of potently carcinogenic polycyclic hydrocarbons with bacteria of phage as target

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CARCINOGENICITY IN POLYC YCLIC HYDROCARBONS

organisms did not reveal strong mutagenic activity. Brookes and Heidelberger [ 161 ] suggested that the binding of 7,12-dimethylbenz [a] anthracene to DNA, demonstrated recently for rat cell in vivo [162], is a necessary but not sufficient condition for carcinogenesis. From photochemical studies, there was no support for a direct relation between carcinogenicity and photochemical (presumed oxidative) binding to DNA [163] ; the effectiveness of anthracene was emphasized . (In the present review, detailed consideration of photo-induced reactions has been excluded since these may be more relevant to skin cancer than internal cancer.) Aromatic hydrocarbons have long been known to interact weakly with, and be solubilized by, purines [ 9 8 ] . The demonstration of carcinogen-DNA cornplexing, as with the binding of 7’12-dimethylbenz[a] anthracene to both replicating and non-replicating DNA [164], led to the notion that such an interaction (especially intercalation, or insertion, of the approximately planar hydrocarbon between nucleic acid bases) leads to modification of the DNA information or chain; if this tendency is inheritable, through mitosis, then the term ‘somatic mutation’ is used (somatic cells are non-reproductive). While subject to some criticism, for example, because of the very limited parallels in potency between mutagenenis and carcinogenesis [ 101, the somatic mutation theory can hardly be said to have been disproved; some points merit emphasis. When reinjected into fresh animals, isolated nucleic acids from polyoma virus particles caused tumours (see references cited in reference [35] ). Coupled with the identification of other cancer nucleic acids, this suggests that a permanent change at the genetic centre of the cell is associated with cancer. However, this was thought to be associated only with a method of growth acceleration, rather than being relevant to initiation or promotion. Other chemically-reactive carcinogens (for example, alkylating agents) are known also to be mutagens; they react with proteins and nucleic acids of the tissue. Although Kotin and Falk [165] concluded that there is an inconstant relationship between the properties of mutagenesis and carcinogenesis, there are experimental problems in testing known mut,Zens for carcinogenicity and vice versa; differences between transport or reaction modes may limit testing of these theories. Recently, DNA has been shown to be the receptor for a metabolite [ 1661 ; specific genetic deletions by carcinogenic hydrocarbons of loci involved in RNA coding have also been proposed [167]. Goshman and Heidelberger [I021 convincingly demonstrated the binding of tritium-labelled hydrocarbons to mouseskin DNA. In addition, following the theories of Mason [80] and of Birks [82], Hoffmann and Ladik [I681 suggested that charge transfer to proteins (such as DNA)

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by interacting polycyclic carcinogens caused chain-pair disruptions and, especially, chain-pair separation. Thus, specific deletions or alterations could occur in DNA chain replication, leading to cancer DNA. By proton magnetic resonance (p.m.r-) spectroscopy, Gerig [ 1691 showed that the large a-chymotrypsin molecule interacted more strongly with carcinogenic D-tryptophan as a reversible inhibitor than did the L-isomer. Following the study of drug binding to bovine serum albumin by Fischer and Jardetzky [I 701 , p.m.r. measurements of this kind are increasingly being made. As mentioned in Section 3.3.1, recent work [I371 implies that the carcinogens and mutagens are the epoxides produced in intracellular metabolism, rather than the polycyclic hydrocarbons themselves. Polycyclic epoxide intermediates, which react with DNA and protein, can be more potent carcinogens than the parent polycyclics. Thus epoxides (and also some phenols) produced by metabolism of methylcholanthrenes and benzanthracenes are mutagenic and cytotoxic to Chinese hamster cells. Mutagenic potential for phage T2 also correlates with carcinogenic potential for epoxides of dibenzanthracenes [ 1401. Evidently, at least for some low-activity polycyclics without active methyl groups, metabolic activation [171], probably via the epoxide as the final derivative [137], is a pre-requisite for macromolecular binding and subsequent mutagenic and carcinogenic behaviour [50, 1391. It is assumed that these epoxides form more rapidly than they are deactivated by further metabolism to hydroxylated derivatives, unless that deactivation is itself responsible for the interference (e.g. crosslinking between or within biomacromolecules) which leads to mutagenicity/carcinogenicity .

4. APPRAISAL OF INTERCONNECTIONS BETWEEN ELECTRONIC THEORIES 4.1. ELECTRONIC ENERGY-LEVEL TRANSITIONS

Mention has already been made (Section 3.3) of correlations between carcinogenicity and electronic transitions in polycyclic hydrocarbons. Figure 4.3 represents the distribution of electrons between levels for several states, while Figure 4.4 indicates the electronic transitions (labelled with proposer’s name) which have been postulated as being involved in charge-transfer mechanisms of carcinogenesis. From ultraviolet absorption spectra, Sung and Lazar [85] not only confirmed experimentally the correlations of Mason, and indirectly of Birks, but also established the validity here of HMO calculations of electronic transition energies. Of the a,& and ,o band frequencies, the p-band is associated with

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CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

electronic transition energy, aE,, a is associated with AE2,and /3 with aE3 (Table 4.7); for alternant hydrocarbons, M.O. theory (see also Chapter 9 in reference [I 51 ) indicates that AE, and AE, should be equivalent. As a corollary of the mathematical MO treatment (Chapter 8 in [172]), theoretical AE,, and AE2 are related, as are M I (HMO) and p [ I 731 . This interdependence of the electronic and associated charge-transfer theories again raises the thought that a non-specific interaction, dependent on hydrophobichydrophilic character and electronic structure, may be ultimately responsible for carcinogenic activity. On the other hand, the lack of any general correlation between carcinogenicity and either ionization potentials or oxidation potentials ('WE1 in HMO calculations) has been stressed [ 1741 . But the proposals of Mason

-+

llolecular orbital ym+2

-

+m+l

-

*m-l

it-

Ground state

- t + -

+t it it

First excited sing1 e t z tate

First excited triplet state

Second excited singlet state

y;,m+l

%ll+l

'm,m+2

Figure 4.3. Diagram o f HMO states in theories of carcinogenesis; orbital (see text)

Molecular s t a t e s

YO

S

is the last occupied

AE2

T r a n s i t i o n used in c o r r e l a t i o n o f ;lason [so] a n d a l s o Hoffman and Ladik [168]

AEi

Transition used in c o r r e l a t i o n o f Birks [82]

AE,

Transition used i n correlation o f S t e e l e , Cusachs and IkGlynn [83]

Figure 4.4. Electronic transitions involved in theories of carcinogenesis

D.W. JONES and R.S. MATTHEWS

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[80] ,Sung and Lazar [ 1731 ,and Birks [82] depend on a parabolic rather than a linear fit of the data (as mentioned in Section3.2) and may still apply for restricted systems; indeed, some such limitation applies to theories on most aspects of carcinogenesis. Thus Cammerata, Yau and Rogers [175] presented relations between ionization potentials-molecular polarizabilities and partition coefficients. The inference of a link between solubility theories and MO indices is not unexpected in view of the influence of molecular polarizability on ‘dispersion-attraction’ interaction energies in solution. An approximate, though rather less relevant, inverse dependence of ionization potential on polarizability has been noted [ 1761 . Table 4.7. CORRELATIONS BETWEEN HMO-CALCULATED ELECTRONIC TRANSITIONS AND FREQUENCIES OF U.V. BANDS [ 8 5 ] Correlation

Correlation coefficient

AE2 (HMO) VS. (Y AE2 (HMO) VS. p AE2 (HMO) VS. (Y, (Y vs. p

0.90 0.93

~~

@

0.95

0.87

4.2. REACTIVITY PARAMETERS AND STRUCTURE

In hydrophobic-lipophilic [ 1771 interactions (which must be related to partition coefficients), adsorption of molecules at specific sites, due to van der Waals forces in the presence of desorbing agents, does not increase linearly with the number of atoms in the molecules [l I ] ;rather, it is the ratio of the total vun der W a d s forces to molecular surface area ( A ) that is significant. The van der Waals forces can be represented by a free-valence index (Fr, ‘left-over n-bonding power’) [178], where the factor Eatom FJA decreases with symmetrical, compact, multi-ring structures. Thus polycyclic hydrocarbons such as the inactive anthanthrene (57) (an exception to the K-L region theory) are sensitive to desorption. Since HMO calculations by Kouteck?, Zahradnik, and CEek [118, 1791, and by Streitwieser ([ 1721 ,Chapter 1 1) show correlations between loculisution energies (usually Lr+) and free valence, superdelocalisabilities, polarisabilities, charge densities, reactivity number ( A , ) and some (Pullman) carcinogenicity indices, the free-valence indices summation may represent just one component in a more general structural correlation. While noting the apparent influence of reactivity indices on carcinogenicity of parent hydrocarbons, Meyer and Bergmann [180] recognise that additional factors will influence the potency

192

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

of methyl-substituted hydrocarbons. Bartle and Jones [ 181] have emphasised the dangers in following apparent correlations between reactivity parameters and, for example, p.m.r. chemical shifts. Correlation of carcinogenicity with ‘molecular encumbrance area’ 11 11 (120 A2 for benz[a] pyrene, for example) is somewhat unsatisfactory because many non-carcinogens have similar encumbrance areas. An apparent correlation with area may be a fortuitous consequence of correlations of other structure-dependent physico-chemical (electronic) parameters with carcinogenicity. For example, in the more general context of defining biological response in terms of partitioning of aromatic molecules between polar and non-polar phases, Rogers and Cammarata [182] found that, of many indices derived from molecular orbital theory, only summations of atom charge density, q , , and atom electrophilic superdelocalisability, S,, gave statistically significant correlation with partition coefficients, P: In P = 0 . 6 6 7 0 c S ,

-

2.5395

c

lqr I + 0.4777

.

(3.1)

(For alternant hydrocarbons, one would expect 14,l to be zero.) The relevance of partition coefficients to theories of internal transport has already been discussed in section 3.2. Since CS, depends on the number, N , of atoms in the structure, so will the partition coefficient, P, as is consistent with experimental measurements of P. The relation between theories of binding areas and of partition coefficients underlines the dangers of discussing different aspects of carcinogenicity separately. Experimentally, Hansch and Fujita [65] found that carcinogenicities ( A ) and partition coefficients (P) of a range of benzanthracenes and benzacridines are related by: log A = -0.14 (log P - 5)’

+ 0.32 (log P - 5) + 28.07 E - 35.26 .

(3.2)

Here e is the total Pullman charge of the K region and, to facilitate curve fitting, the +, ++, etc. activities were transposed t o a numerical scale. Franke 1741 considers that the squared term in Equation (3.2) is of doubtful significance (it leads to predictions of erroneously low activities for 7~2-dimethylbenz[ a ] anthracene and for hexacyclic hydrocarbons); log P may be inadequate as a measure of the experimental hydrophobic binding coefficient, log K,, used by Franke. For carcinogenic potency, the Pullman complex index, I (sometimes called Zp), where

OLE = BLE + CLE (min)

(3.3)

D.W. JONES and R.S. MATTHEWS

193

must be under 3.31 p. A French team [I831 proposed the electrophilic localisation energy, A , , , of the K region as an index; it is defined as the n-electron energy difference of the parent hydrocarbon and the torso remaining after removing the ortho-p, orbitals of a bond (which torso contains three electrons less than the parent). For potency, A , , < 3.64 0. Effectively,

A,, = BLE (or A,) + E

(3.4)

where e is the energy of the highest occupied bonding orbital of the torso; a parallel behaviour of OLE and A,, is not favoured [118]. It should be noted that the A , , index incorporates features both of Pullman indices and of the energy-transition correlations. (In even alternant aromatic hydrocarbons, which have MO's symmetric about the non-bonding basis in the torso molecule, 2 E = AE,.)

The correlation observed by Dipple, Lawley and Brookes [ 1311 between potency and the coefficient (a,,) of the non-bonding molecular orbital (NBMO) of a carbonium-ion residue from the enzymic hydroxylation of a K-region in a carcinogen is not unexpected. Following Dewar and Sampson [184], the stability of the carbonium ion may be defined as I-a,,; increased potency is associated with increased MO-calculated stability of the 'IT systems of carbonium-ions (e.g. 6 5 , alternatively represented as 66 for simple HMO calculation) derived from a series of parent aromatic hydrocarbons. In odd-alternant hydrocarbons, Dewar [ 1851 used similar NBMO coefficients (a,) for calculating localisation energies at a single atom; elimination of the 2p, orbital permits calculation of the conjugation energy of that atom to yield a chemical-reactivity number, Nr = XaOs + a,,)

.

(3.5) Atoms s and r are two bonding positions of the specified atom. Streitwieser ([172], Chapter 11) shows a correlation of localisation energies (cation-L,t) with Nr. By comparing results from K regions only, Kouteckq and Zahradnik [ 1181 also noted the partial interdependences of these parameters for particular categories of carbon atoms. Further, allowing that a,, contributes more to N , than does a,, ([172], Chapter 1 l ) , the partial correlation betweenN, and l-aor is established. Thus, as Dipple et al. hinted [ 1311 ,their and the Pullman correla-

194

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

tions are interrelated. It must be noted that, although localisation energies calculated rigorously by Wheland’s method do not coincide with those by Dewar’s more approximate method, the results are parallel and they provide a reference curve [45] . While Scribner [ 1861 expresses surprise that the Dipple correlation (1-uor > 0.28 for potency) is so successful in predictions, Pullman, Umans and Maigret [ 1471 criticize the correlation, especially in the apparently unjustified use of uor only. Now the precursor to which the calculation applies can be written in two ways (of which 67 is not the less important biologically), so that this criticism is perhaps valid only in part. As the search for the crucial aspect of electronic structure continues, hydrophobic-lipophilic forces may be expected to play an increasingly significant role. While hydrophobic bonding may have only secondary influence (compared with chemical reactivity) on carcinogenesis, it seems particularly promising in explaining the inhibitory action of inactive and weakly active hydrocarbons on the potency of strongly active carcinogens [74]. 4.3.AROMATICITY AND RlNG CURRENTS: NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

In view of the links that lipid character may be expected to have with both carcinogenicity (via solubilizing ability) and aromaticity, induced n-electron ring currents [ 1871 - a manifestation of diamagnetic anisotropy - should link with carcinogenicity. High-resolution proton magnetic resonance (p.m .r.) spectra of polycyclics [ I881 are capable of yielding two kinds of parameter, the chemical shifts (6) and the spin-spin coupling constants (4,which depend on the magnetic (and hence geometrical and electronic) environment of the magnetic nuclei, and hence also on interactions with other molecules. With their aid, evidence about aromaticity can be deduced in several ways [189]. In some heterocyclic isosteric analogues of polycyclics, such as benzo [ b ]-thiophen and dibenzothiophen, the heterocyclic rings carry much the same current as in benzene [ 1501 . However, currents in the thiophenoia I I I I ~ Sor acenaphtho [ 1,2-b] (68) and acenaphtho [ 1,2-c]thiophen (69) are smaller than in the benzenoid ring of fluoranthrene (70) [191]. Complexity of p.m.r. spectra from closely coupled,

D.W. JONES and R.S.MATTHEWS

195

often overlapping, spin systems in polycyclic hydrocarbons [ 1881 dictates the desirability of recording the spectra at high fields [192] in order that they can be solved. Knowledge of v and J can enable the application of molecular-orbital theory to polycyclics to be checked; I 3 C resonance may be useful in the future. Coupling constants often show approximately linear relations [ 1931 with HMO a-bond order of the intervening bonds. Since relations have also been proposed between n-bond order and bond length, some association might be expected between high resolution p.m .r. ortho-coupling constant (measured in solution) and short bond length (measured in the crystal), and possibly also with high carcinogenic activity. While there is some substance in this for the more planar molecules, Bartle, Jones and Matthews [ 1941 conclude that neither coupling constant nor bond length at the K region is simply related to carcinogenicity. While the relatively large differences in n-bond order calculated from ortho-coupling constants for K regions of potent carcinogens (for example, benz [a] pyrene and 7J2-dimethylbenz [a] anthracene) do not necessarily undermine the concept of Pullman indices, they suggest a limit to the influence of C-C bond geometry at the K region. For sterically hindered polycyclics, chemical shifts can be indicative of deviations from planarity [195]. A major contribution to these shifts appears to come from n-electron ring currents calculable from MO theory [187, 1961. Following earlier calculations on carcinogens such as benz [a] pyrene, benz [a] anthracene [197], and dibenz[a,h] anthracene [198], Mallion [I991 has found that n-electron ring currents in heptacyclic carcinogens are not very different from those in smaller analogous carcinogens. Small deviations from chemical shifts expected from LCAO calculations in benz [a] anthracene derivatives may reflect an association of carcinogenic activity with the stereochemistry of the C-12 substituent in the L region and also possibly of a geometric factor in the K region [200] . For ‘bay’ hydrogens [I951 confronted across the bay by methyl groups, as in 7-methyl- and 7,12-dimethylbenz[a] -anthracene, displacement of rings and/or methyl groups can concurrently influence carcinogenicity and reduce ringcurrent and bond-anisotropy contributions to the chemical shifts [ 1921 . 5. CONCLUSIONS

For elucidation of the molecular mechanism of hydrocarbon carcinogenesis, a prerequisite is knowledge of the cellular mechanism, for which there are three main possibilities [201]. Hydrocarbon activation of a latent oncogenic virus is undermined by the observation that hydrocarbon-induced sarcomas are anti-

196

CARCINOGENICITY IN POLYCYCLIC HYDROCARBONS

genic, the antigens being individualistic [202] . Secondly, selection of pre-existing ‘clones’ of malignant cells by the hydrocarbon [203] seems to be ruled out by in vifro experiments on mice tissues [204], provided it is accepted [I711 that the model is valid for the situation in vivo. Thirdly, if the hydrocarbon transforms normal cells to cancer cells directly, as seems most probable, this can be achieved via either (or both) of two kinds of mechanism. These are (1) somatic gene mutation resulting from hydrocarbon binding to DNA and so altering its structure (genetic change); and ( 2 ) permanent metabolic (non-mutational or extra-genetic) modification of the gene expression, which can still lead to perpetuated changes [203]. Disruption of cellular growth control mechanisms, w h c h underlies the carcinogenicity of aromatic hydrocarbons, can be achieved by quite small changes in cellular constituents. It is not surprising, therefore, that the physico-chemical characteristics (structural, experimental, theoretical) or many individual hydrocarbons have been investigated in an effort to discern the features which distinguish carcinogenic from non-carcinogenic hydrocarbons; the very extent of the literature means that it is difficult to survey it without taking a (slightly biassed) standpoint. As a result, a considerable number of tentative interconnected empirical relations have been deduced. One explanation of their approximate nature is that the parameters chosen are insufficiently accurate over a wide range of structures with varying carcinogenicity. Further, the biological screening data are of restricted accuracy [205] although this limitation is largely removed if attention is confined to a series of wide carcinogenic range, such as the benz[a] anthracenes (Section 2.1), which have been screened by several workers and are likely to adopt a common mechanism. (The remarkable paucity of recent activity data for alternants and, a fortiori, non-alternants should be corrected; there would also seem to be a need for a reassessment of activity data already available.) Another view is that the complexity of the problem renders essentially monoparametric iimestigations misleading. Conversely, apparent improvements in correlation coefficients, r, attainable when a multiparametric treatment replaces a series of monoparametric ones must be treated with reserve [74]. If both the above views are set aside, it appears that the notion of the responsibility of a single mechanism for hydrocarbon carcinogenesis must be replaced by two possibilities: (a) responsibility of one molecule for initiating a specific cytoplasmic event leading to disruption of cellular metabolism ; or (b) occurrence of more general disruption via a multicellular process, i.e. a set of events generated from a distribution of interacting events within the cell. To distinguish between (a) and (b) in vivo would be difficult. Investigation of the target-area side of the problem, i.e. the growth mechanism or metabolic function disrupted by aromatic carcinogens, may lead to the

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possibility of curing cancer by controlling development. On the other hand, knowledge of the processes governing chemical initiation (transport and activation) by aromatic hydrocarbons may ultimately lead to cancer prevention via either desensitisation of cell replication machinery or elimination of active molecules from their target areas. At the opening of the 1968 Jerusalem Symposium on Physico-chemical Mechanisms of Carcinogenesis, the Pullmans suggested [ 101 that the principal (interrelated) problems of controversy were: (i) the nature of the principal receptor, whether protein or nucleic acid (and, if the latter, whether DNA or RNA’s); (ii) the nature (physical or chemical) of the interaction or reaction; (iii) the nature (initial molecule or metabolite) of the proximate carcinogen; and (iv) the relation between carcinogenesis and mutagenesis. Colburn and Boutwell [206] concluded that at least there does seem to be a positive correlation of tumorigenesis with one or both of the nucleic acids DNA and RNA. Very recently, it was stated that the critical experiments have yet to be done to ascertain which (if any) of the macromolecules to which hydrocarbons (or their expoxides) bind is the primary target that triggers the carcinogenic process [ 1391 . From quantitative structure-activity analysis, Franke [207] suggests that microsomal arylhydrocarbon benzopyrene hydroxylase may be the first step in chemical carcinogenesis of polycyclic hydrocarbons. While some progress has been made subsequently towards solution of all these questions, especially in connection with metabolic products, it cannot be said that the answer to any is known with certainty. (Some of the recent work [208] on metabolites of polycyclics is more relevant to excretory than initiation processes and has not been reviewed in detail; admittedly, ease of elimination of a polycyclic or its metabolite from the system will influence its carcinogenicity.) Indeed, despite the accumulation of many data on the interaction with cell components, it is sombre to report how slow have been the advances in the mechanism of hydrocarbon carcinogenesis. With the combination of several techniques, the study of cellular and molecular biology is passing from the essentially descriptive state to one that can yield an understanding of specific interactions of biological processes. The time may well be approaching when cellular and molecular descriptions of hydrocarbon carcinogenesis can be achieved, even if one cannot hope yet to provide unequivocal chemical criteria of carcinogenic potency.

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