The soothing-noise industry

Fd Cosmet. Toxicol.

Vol. 4, pp. 525-537. Pergamon Press 1966. Printed in Great Britain

ARTICLES OF GENERAL INTEREST THE SOOTHING-NOISE INDUSTRY The production of soothing noises is the USA's fastest growing industry. It flourishes thanks to public clamour, stimulated by highly vocal do-gooders, honest-pennY-turning journalists and authors of marrow-curdling books on the dangers of chemicals. Its impetus is provided by the needs of Senators for causes calculated to yield publicity and to ensure re-election. Its fires are most directly stoked by the FDA, with the hot breath of Congress down their necks, the hounds of the Press at their heels, and scalpels poised throughout the world to dissect every pronouncement, every decision and every attempt at effective action. The products of the soothing-noise industry come in the form of Reports. Thanks to the influences enumerated above, the Reports grow thicker and thicker, and many many more of them are required for each new compound, be it drug or food additive. Battalions of scientists and technicians, cohorts of automated auto-analyzers and computerized statisticians, regiments of typists and co-ordinators, countless myriads of rats, mice, dogs, pigs and other animals devote their existence to the selfless pursuit of the final objective: the Soothing Noise Report (SNR). Stony-hearted men who would think, not twice, but fifty times before contributing a penny piece to toxicological research, unhesitatingly dig deep in their pockets to pay for SNR's. Finally there comes the proud moment when photographs are taken to record for all posterity the puniness of man when placed next to the massive piles of the SNR's needed for only one new product. The magnus opus is complete. At the receiving end, a quiet radiance glows. Leafing eagerly through the faultlessly reproduced and meticulously assembled pages of data, one soon comes upon the glad tidings: two years in rats produced no adverse effect. A few deft flicks of the wrist and, hey presto--two years in dogs produced nothing either. Was the hint taken to inject as well as feed ? Yes, it certainly was and, bully for them, nothing happened. What about mice--no stinting of those ? Never fear, there were hundreds upon hundreds of mice, kept to a ripe old murine equivalent of three score years and ten. Apart from aging, nothing happened. As for the five-generation test, the no-sparing of species in seeking out teratogenesis, the searches among fruit-flies and yeasts and bacteria for mutagenesis--nothing happened. By this time radiance has given place to ecstasy and ecstasy has reached its zenith. The epicurean repast needs only those subtle touches provided by six months in monkeys, ninety days in rabbits, some special work in guinea-pigs, hamsters and cats--all, of course, establishing that nothing happened. In some circles it is regarded as legitimate to add a subtle touch or two, illustrating species differences: a spell of giddiness in the nine-banded armadillo (naturally, only at the highest dose of test compound), halitosis after meals in the electric eel. Over such results heads are shaken and the conclusion drawn that they are probably not applicable to man. In this way it can truly be said that difficult problems have been faced up to and real scientific decisions arrived at. A good job has been well done. Where is the ingrate who would question the value of this immense effort, or the devotion to duty displayed throughout the execution of this vast enterprise ? SNR's are meant to soothe, not to illuminate. What if nothing is known about the fate of the compound in the 525

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human body? SNR's are meant to satisfy, not clarify. What if the pharmacodynamics haven't been thought of? SNR's are meant to pacify, not stimulate enquiry. What if the mode of action is obscure? SNR's are meant to tranquillize . . . hypnotize . . . and finally to anaesthetize . . . Research is out, man, way way out. FDA's new Commissioner, Dr. James L. Goddard, must have had SNR's in mind when he said to Toxicologists recently: "Past and present thinking about animal studies-numbers and time in particular--are, I submit, not good enough for any of us. Are we trapped in a numbers game ?" The route of escape from the numbers game is clear. Encourage the laboratories in this field to carry out the selective, specific and sensitive toxicological studies that the particular problem demands; in other words, the sort of studies they'd like to d o - - b u t the boss or client won't pay for at present because this is not what the authorities ask for. Also, let us recognize frankly the woeful lack of suitable methods for detecting the response to exposure to a compound and for differentiating exposure from injury. Our state of backwardness will continue as long as most of the funds, talents and time are devoted to playing the numbers game with SNR's as counters.

R U N N I N G N E C K AND N E C K ON C R O T O N OIL Two teams of workers led by Hecker in Heidelberg and by Van Duuren in New York are actively engaged in the determination of the nature and biological activity of the active principles of the tumour promoter, croton oil. Hecker and his colleagues have already presented some of their findings in several preliminary communications (Cited in F.C.T. 1965, 3, 339) which are now superseded by full reports of this work. Hecker et al(Z. Krebsforsch. 1965, 66, 478) furnish details of the countercurrent distribution and chromatographic methods employed to isolate the two active components of croton oil, which are referred to as compounds A and B. The acute toxicity and the irritant and cocarcinogenic activities of croton oil are almost wholly accounted for by these two compounds, which are present in a combined concentration of less than 1 ~o and are claimed to be the most potent cocarcinogens yet discovered. B proved to be more active than A, producing sarcomas and leukaemias in addition to the carcinomas induced by either compound in animals pretreated with subliminal doses of the carcinogen, 7,12dimethylbenz[a]anthracene. A further paper in the series (Hecker & Bresch, Z. Natu~ 1965, 20b, 216) gradually unveils the identity of the A1 component, as a diterpenoid alcohol of molecular formula C20H2806 containing the substituents previously reported (Cited in F.C.T. 1964, 2, 517). What originally was thought to be one compound has now emerged as a family of seven. First compound B was resolved into B1, B2 and BX (ibid 1965, 3, 339). B1 (C37H5808) and B2 (C35H5408) turned out to be the diesters of the parent alcohol of compound A1 with S-2-methylbutyric acid and lauric (B 1) and capric acids (B2) respectively (Hecker & Kubinyi, Z. Krebsforsch. 1965, 67, 176). And no longer are we left in the dark over BX. No less than five irritant and cocarcinogenic components (B3-B7) have so far been detected (Clarke & Hecker, ibid 1965, 67, 192). In common with their predecessors, these compounds also have the diterpene alcohol C20H2806 (now called phorbol) as their basic unit, being diesters with

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one short chain fatty acid (acetic, S-2-methylbutyric and tiglic) and one long chain fatty acid (octanoic, decanoic and dodecanoic). Persevering still further, Hecker et al. (Angew Chem. (Int. Engl. Edit.) 1965, 4, 1072) have found that of the three esterifiable hydroxyl groups of phorbol, the primary one on C-20 is free in the isolated compounds; the secondary one on C-5 and the tertia~ one on C-8 carry a short- and a long-chain fatty acid residue respectively• A structure of phorbol is proposed (Fig. 1) with the possible variations in substituents also indicated. Running neck and neck with Hecker and his co-workers into the final straight of this marathon come Van Duuren & Orris (Cancer Res. 1965, 25, 1871). Starting from croton seed, details are given of the extraction and isolation of the two cocarcinogenic factors A and C, previously referred to by these workers (Cited in F.C.T. 1965, 3, 340). Even at such low doses as 0.5 #g applied twice weekly, A and C demonstrated their tumour-promoting propensities following pretreatment with 7,12-dimethylbenz[a]anthracene. A and C, which constitute approximately 1 ~o of the whole croton seed extract, provide almost the total tumour-promoting activity of the extract. There is still some way to go before the complete structural determinations of A and C have been clinched. No new light is thrown on the fatty acids obtained on alkaline hydrolysis of A and C. The present claim is that A and C do not contain an aromatic nucleus but are esters of phorbol, C20H2806, a known component of croton oil which the present authors took the trouble to isolate from croton resin. It is noteworthy that the semisynthetic compound derived from the resin, phorbol myristate acetate showed notable tumour-promoting activity. Phorbol acetate and a hydrogenated polyphenolic ester showed no activity.

H3C

20 CH~OH

~

H

Ro

c.,

/\

__OH

CHs CH3 R--RI=H

R--H, R ~ A c e t y l ; O-acetylated at C-20 R--Decanoyl, Rl--Acetyl R--Lauroyl, R l--Acetyl R~Myristoyl, RI--Acetyl R--Acetyl, R I c H R--Acetyl, Rl~Decanoyl R--Acetyl, Rl~Lauroyl R~Acetyl, Rl~Myristoyl

FIG. 1. Proposed structure of phorbol with possible substituents yielding probable active components of croton oil.

[So the croton oil saga continues and there is little to choose between the two groups of hardy warriors battling their way to the bitter end. If similar efforts were directed to but a minute proportion of the galaxy of natural materials masquerading in the vegetable kingdom, the possibility of permitted lists for natural foods might well become a reality. And why not indeed !]

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DON'T OVER-INDULGE IN AMINO ACIDS Although vital for normal body function, amino acids have been shown to produce deleterious effects when given at excessive dietary levels to experimental animals. Thus too much methionine (I) causes reduced food intake, weight loss, organ damage, increased urinary excretion of amino acids and alterations in liver enzyme activities. Some of these effects are alleviated by supplemental.glycine (II) or arginine (III) (Cited in F.C.T. 1964, 2, 137 & 281). Our knowledge of the adverse effects of excess dietary amino acids, especially I, has been brought up to date in two short reviews on the subject (Nutrition Reviews 1965, 23, 202 & 317). Notable contributions in this field are being made by Klavins and co-workers. For example, carefully-controlled feeding experiments by Klavins & Peacocke (Br. J. exp. Path. 1964, 45, 533) have shown that some of the changes induced by I at about 4-5 Yo of the diet are caused by weight loss but others are more specific toxic effects of I. These specific changes include increased liver weight and iron (Fe) deposition in the Kupffer cells, Fe deposition in the spleen, pancreatic damage and haemolytic changes, including decreased red cell and increased reticulocyte counts. Although the addition of equimolar amounts of II and/or III to the diet did not prevent these changes, II reduced the weight loss, both H and III partially reduced Fe deposition in the spleen (but not in liver) and III caused some increase in the depressed red cell count. The authors suggest that alleviation of some of these changes by II and/or III is due to altered absorption and/or excretion of I, but this possibility has not been investigated. The second review features further work by Klavins & Johansen (Archs Path. 1965, 79, 600) which compared the effects of excess dietary intake of I and homocystine (IV), an intermediary metabolite of I. Although many of the changes were similar, striking differences were found in the distribution pattern and amount of Fe deposited in the liver (more with IV), in the Fe deposition in the spleen (none with IV) and in epiphyseal changes (induced by IV). Moreover, it was found that whereas serine (V) alleviated or prevented all the effects of IV (probably by combining with IV to form cystathione), it had no effect on the changes induced by I. This certainly suggests that I and IV act differently and that the IV moiety of I is not entirely responsible for the changes induced by I. It was also shown that the methyl moiety of I is not important in Fe deposition in the liver and spleen, since these changes were not produced by compounds with labile methyl groups, such as choline (VI). Dietary excess of VI, too, is deleterious, causing kidney lesions (deposition of Fe and calcium in the tubules), which were prevented by both I and IV. These results show that interactions between I, IV, V and VI prevent some of their individual toxic effects but, particularly in the case of excess I, tissue damage is not completely prevented. The mechanisms of action and interaction are not yet understood. Phenylalanine (VII) is another amino acid which assumes considerable importance in certain individuals. A genetically-determined enzyme defect in the ability to metabolize VII to tyrosine is responsible for phenylketonuria, a serious inborn error of metabolism resulting in mental deficiency (Cited in F.C.T. 1966, 4, 449). In young rats the toxic action of excess dietary VII has been attributed to the formation of tyrosine owing to the similarity in the toxic effects exercised by each compound (Dolan & Godin, Can. J. Biochem. Physiol. 1966, 44, 143). Toxicity is manifested by eye lesions and swelling of the toe and bladder and by obstruction of the ureter and urethra by crystals of the amino acids. In a study by Thompson & Kano (J. Psychiat. Res. 1965, 3, 91), pregnant rats were fed diets containing 3 70 VII and 3 70 tyrosine. Although the metabolism of VII was specifically

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affected in the mothers (excretion of phenylpyruvic acid), this was not evident in the offspring. However, the behaviour of the offspring was definitely affected although various tests (including walking through mazes) showed that it was temperament rather than intelligence that was affected. Previous work on rats has indicated that post-weaning rather than preweaning treatment with VII is necessary to affect intelligence, and L~af et al. (Nature, Lond. 1965, 208, 1021) found that behavioural disturbances (lever depressing perform'ance) in the offspring of rats fed diets containing 7 70 VII were much more pronounced when VII was also fed after weaning, although in this case post-weaning treatment alone was ineffective. It is well known that in human phenylketonurics brain damage is not apparent during the first few months of life. Another inborn error of metabolism in a small number of individuals causes the excessive build-up of II (hyperglycinaemia). So far the specific defect in the enzymes responsible for the metabolism of II has not been discovered (Nutrition Reviews 1965, 23, 191). The intermediary conversion of II to V is inhibited in the hyperglycinaemic patient, although metabolism of V proceeds normally. Since several pathways of I I ~ V are said to exist, more than one defect could be present. It has also been suggested that the removal of II from the blood may require metabolic conversion to V.

IT'S THOSE NITROSAMINES A G A I N Introduction A large number of nitrosamines (NA) are now known to be powerful carcinogens. Their detection in tobacco smoke (Cited in F.C.T. 1963, 1, 253; ibm 1965, 3, 501) and in meal from herrings preserved with nitrite (ibid 1965, 3, 525) indicates that they must now be regarded as potential environmental hazards. The great interest in NA is reflected in the large number of publications that have appeared since our last review of this subject (ibm 1965, 3, 498). Some of these are summarized below. Tumour production The remarkable capacity of NA to produce a great variety of tumours at different sites is well illustrated by the effects of long-term oral administration of dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) to mice (Takayama & Oota, Gann 1965, 56, 189). Moreover, there were important strain differences in the susceptibility of mice to these two carcinogens. Other studies have now demonstrated the hepatocarcinogenicity of D E N in rabbits (Rapp et al. J. hath. Cancer Inst. 1965, 34, 453; Schm~ihl & Thomas, Naturwissenschaften 1965, 52, 165) and monkeys (O'Gara & Kelly, Proc. Am. Ass. Cancer Res. 1965, 6, 50), these two species thus sharing the company of the rat, mouse, hamster, guinea-pig, dog and trout in this respect. The type of liver tumour produced varies in different species and a study of hepatic haemangioendothelioma induced in mice by D E N has shown that on reducing the dietary intake of DEN, the total dose required to induce tumours also decreases, indicating an "acceleration" process (Schm~ihl & Thomas, Z. Krebsforsch. 1965, 66, 533). Tumour induction is said to follow the general formula d x t " = K , where d represents the daily dose, t the latent period of tumour induction, n a constant lying between 2 and 3, and K another constant based on a 50 70 incidence of tumour yield. The site of action is unpredictable and does not seem to depend on chemical structure. For example, skin carcinomas have now been induced by repeated intravenous injection

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of diazoacetic ester into rats (Druckrey et al. Z. Krebsforsch. 1965, 66, 523), and tumours of the brain and spinal cord have been selectively induced in rats by administering Nmonomethyl-N-nitrosourea by the same route (Druckrey et al. ibid 1965, 66, 389). In contrast, the carcinogenic action of N,N',N'-trimethylnitrosourea after oral dosing is directed more at the peripheral than at the central nervous system (Ivankovi6 et al. ibid 1965, 66: 541). NA frequently produce tumours of the nasal cavity and carcinomas have recently been induced in this location by a variety of alkylnitroso compounds including DMN and DEN, following administration by various routes to rats (Thomas, ibid 1965, 67, 1). More interesting, however, is the finding that histologicallythese tumours, like oesophageal tumours (Cited in F.C.T. 1964, 2, 63), are very similar to those occurring in man. Maternal transfer to foetus or child Maternal transfer of NA has been demonstrated in hamsters and rats (ibid 1965, 3, 499). In mice too, DEN is transferred to the foetus when administered during pregnancy, as shown by a significant increase in the incidence of lung tumours accompanied occasionally by liver tumour development (Mohr & Althoff, Z. Krebsforsch. 1965, 67, 152). The maternal transfer of DEN in hamsters with subsequent development of fiver necrosis and tracheal tumours in the young has been confirmed (Mohr et aL ibid 1965, 66, 536). In order to determine whether maternal transfer proceeds exclusively through the placenta or in addition via the milk, newborn hamsters from mothers receiving DEN during the second half of pregnancy were suckled by untreated mothers, and offspring of untreated mothers by DENtreated ones (Molar & Althoff, Z. Naturf 1965, 20b, 501). Since tracheal tumours only developed in the former group, transmission of DEN in the milk appears to be ruled out. DEN has also been detected in hamster embryos after intracardiac injection into the mother, although none could be found in maternal liver or placenta or in the embryos when it was administered orally, subcutaneously or intraperitoneally, due no doubt to its slow absorption, dilution in maternal tissues and the formation of metabolic products (Mohr et al. Naturwissenschaften 1965, 52, 188). Damage at the cellular level A new method of approach has been to study the site and mode of action of N-methylN-nitrosourethane in single cells, namely in Amoeba proteus (Ord, Nature, Lond. 1965, 206, 413). On exposing amoebae to low non-lethal concentrations of this carcinogen, the first noticeable effect was a delay in cell division which was followed by abnormalities in cell size and deranged nuclear and cell division. Both nuclei and cytoplasm appeared to be affected, as shown by experiments in which nuclei were transferred between control and treated amoebae and these effects were carried through several generations before disappearing. Mutant strains also arose occasionally. These studies are stiU only in a preliminary stage but may well provide some insight into the mechanism of toxic action of NA in higher animals. Biochemical mechanism of toxic action A plausible biochemical mechanism of NA action involves conversion in the body into active alkylating intermediates, notably diazoalkane or the corresponding carbonium ion (Cited in F.C.T. 1964, 2, 62). The possibility that alkylation of nucleic acids may be important in the induction of cancer is strongly supported by alkylation of the N-7 position of the

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guanine component of nucleic acids by DMN and DEN in vivo (ibid 1964, 2, 241) and by diazomethane in vitro (ibid 1965, 3, 500). There are, however, certain difficulties in accepting alkylation of nucleic acids as of prime importance in tumour induction by NA. For example, although liver deoxyribonucleic acid (DNA) is methylated in the guanine residues following a sirigle intravenous dose of [14C]DMN (ibid 1964, 2, 241), repeated dosing for several weeks is required to'induce liver cancer. Moreover, the activity of the liver enzyme responsible for the demethylation of DMN, which is an essential preliminary to the formation of the alkylating intermediate and to DNA methylation, decreases during hepatocarcinogenesis. Hence, the importance of establishing that DNA methylation does in fact occur during hepatocarcinogenesis, a.nd this has now been demonstrated by Craddock & Magee (Biochim. biophys. Acta. 1965, 95, 677). After feeding unlabelled DMN to rats for 23 wk [~4C]DMN was injected and the methylation of liver DNA was followed. The initial methylation of DNA was superseded by a loss of methyl groups and it is suggested that this might be due to rejection of the abnormal base methyl guanine. A more serious drawback to this otherwise attractive hypothesis is that several alkylating agents, including dimethylsulphate (DMS), are known to alkylate DNA in vitro but are only very weak carcinogens. For this reason, Swarm et al. (Fedn. eur. biochem. Soc. 2nd Meet. Vienna, 1965, Abstract e73) have compared the degree of in vivo methylation of nucleic acids in various organs of the rat after an intravenous dose of DMS and the powerful carcinogen N-nitrosomethylurea, each labelled with 14C. The degree of guanine methylation was much lower with DMS than with the nitroso compound. These findings support the idea that alkylation of DNA is importaht in NA-induced carcinogenesis and that an alkylating breakdown product is the proximal carcinogen. Possible alkylating intermediates Apart from diazoalkane and the corresponding carbonium ion, the possibility of other active intermediates such as nitrous acid, oxides of nitrogen and hydrazines cannot be excluded. An appraisal of the situation is given in a review by Magee (in Alkylierend wirkende Verbindungen, First Conference on N-Nitroso-Compounds and Lactones, Hamburg, 1963). Nitrous acid comes in as a rank outsider owing to its mutagenic potential (Cited in F.C.T. 1964, 2, 62) but falls at the first hurdle--various tests for carcinogenicity proving negative (Druckrey et al. Arzneimittel-Forsch. 1963, 13, 320). It also deaminates nucleic acids in vitro with the formation of xanthine and hypoxanthine, but in DMN-treated rats this reaction has now been shown to be negligible (Craddock & Magee, Fedn eur. biochem. Soc. 2nd Meet. Vienna, 1965, Abstract e74). Nor are nitrogen oxides carcinogenic (Druckrey & Preussmann, Naturwissenschaften 1962, 49, 498) and this has recently been confirmed by Henschler & Ross (Arch. exp. Path. Pharmak. 1965, 250, 256), who found no increase in the incidence of lung tumours in mice following repeated exposure to 40 ppm nitrogen dioxide for up to 1.5 yr. In addition there is no evidence that occupational exposure to 5 ppm (the maximal permissible concentration) induces lung cancer in man. [Although the fact is not relevant to NA metabolism, readers are reminded that small amounts of NA can be formed in cigarette smoke by the reaction of oxides of nitrogen with amines (Cited in F.C.T. 1963, 1, 253; ibid 1965, 3, 501).] With regard to hydrazines, some are known to be carcinogens (ibid 1966, 4, 192). The powerful carcinogen, N-nitrosoaminomorpholine, has been shown to be converted anaerobically in vitro by guinea-pig liver homogenates into the hydrazine N-aminomorpholine

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(morpholylhydrazine), a reaction requiring reduced nicotinamide-adenine dinucleotide phosphate (Siiss, Z. Natur.f 1965, 20b, 714). Unfortunately neither the in vivo significance of this reaction nor the possible carcinogenicity of this hydrazine has been examined. This hydrazine is not a powerful mutagen for Escherichia coli, although dimethylhydrazine is (Lingens, ibm 1964, 19b, 151). Much more work needs to be done before it will be possible to assess the possible importance of hydrazine intermediates in NA-induced cancer. Dimethylhydrazine, however, which could be a metabolite of DMN, is not carcinogenic (Ross & Henschler, Naturwissenschaften 1963, 50, 503). All in all, the most likely alkylating intermediates in NA mutagenesis and carcinogenesis are the diazoalkanes (Cited in F.C.T. 1965, 3, 500) and such intermediates are probably also responsible for the acute hepatotoxicity of NA. This hypothesis is supported by the finding that aminoacetonitrile (AAN) prevents DMN-induced liver necrosis by inhibiting the enzymic demethylation of DMN (Fiume & Roffia, Nature, Lond. 1965, 206, 1157), since significantly higher liver DMN concentrations were found in the livers of rats pretreated with AAN than in those receiving DMN alone.

THE UBIQUITY OF LEAD Introduction As a consequence of the distribution of lead (Pb) throughout the earth's crust, its presence in food and water and its use in countless manufacturing processes, as well as atmospheric pollution from industrial plants and the exhaust of motor vehicles burning leaded petrol, everyone is exposed to some extent to this toxic metal (Cited in F.C.T. 1964, 2, 606). However, thanks to effective control of the industrial environment, the most severe forms of Pb poisoning such as encephalopathy and palsy are now very rare, Nevertheless in shipbreaking, where fumes from anticorrosive paints are produced in confined spaces and are difficult to control, the hazard is still considerable (ibid 1964, 2, 608). For this reason attention is now being focused on the detection of incipient Pb poisoning in its subclinical stages when the industrial worker, although unwell, is disinclined to seek medical advice. Diagnostic criteria of Pb exposure The Lancet (1966, i, 191) has stressed the importance of early diagnosis. This can usually be achieved by determining the concentrations of blood haemoglobin and Pb and of urinary Pb, coproporphyrin and the porphyrin precursor 5-aminolaevulinic acid (ALA). Test doses of chelating agents such as EDTA and penicillamine may also yield useful information. These rapidly remove loosely-bound extracellular Pb although firmly-bound intracellular metal is only removed very slowly (Castellino & Aloj, Br. J. ind. Med. 1965, 22, 172). Urinary porphyrin excretion shows promise of providing useful criteria of Pb exposure (Cited in F.C.T. 1964, 2, 425) and Cramrr & Selander (Br. J. ind. Med. 1965, 22, 311) have measured the above urinary parameters in shipbreakers who displayed a range of slight to serious symptoms of Pb poisoning. The severity of the symptoms was found to correlate most closely with the excretion of ALA but correlation with urinary Pb and coproporphyrin was poor. Urinary ALA was also found to be the most useful diagnostic parameter following penicillamine treatment, suggesting that the initial excretion of ALA indicates the size of the metabolically-active Pb pool.

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In another article (Lancet 1965, ii, 26) attention is drawn to the resemblance between Pb poisoning and acute intermittent porphyria. Dagg et al. (Q. Jl Med. 1965, 34, 163) made a comparative clinical, biochemical and pathological examination in groups of 50 patients suffering from these two diseases. Anaemia was present in nearly all patients with Pb poisoning, but almost completely absent in patients with porphyria. In addition, whereas paresis in Pb poisoning exclusively affected lower-motor neurones, 10 Yo of the cases with porphyria had upper-motor neurone involvement. The clinical similarity was matched by the pathological and biochemical features. In both conditions, urinary excretion of ALA was pronounced and there was an increased excretion of porphobilinogen, a later precursor of porphyrin than ALA (less marked in Pb poisoning) and coproporphyrin (more marked in Pb poisoning). These workers also compared experimental Pb poisoning and allylisopropylacetamide-induced porphyria in rabbits. Whereas in porphyria, as is already known, the liver was the site of deranged porphyrin metabolism, in Pb poisoning the bone marrow was the main site of attack. Evidence was also obtained that Pb may have a direct neurotoxic action and that anaemia in Pb poisoning is caused by a haemolytic process as well as by direct inhibition of haemoglobin formation in bone marrow.

Sources of Pb intoxication Although, with the exception of shipbreaking, Pb poisoning is no longer a serious occupational hazard, there is still considerable concern about the danger of environmental Pb to children. Many serious and a few fatal cases of Pb intoxication have occurred in young children (Cited in F.C.T. 1964, 2, 609; ibid 1965, 3, 660). The most common source of Pb is exterior paintwork of the flaking off of interior paintwork in old homes. Pica accounts for ingestion of paint. It is thus disconcerting to read that, despite recent advances in paint technology (ibid 1964, 2, 275) Pb is still disseminated by decorations in the USA. It is reported by Schucker et al. (PubL Hlth Rep., Wash. 1965, 80, 969) that in Baltimore between 1956 and 1964 abnormally high Pb absorption was detected in 1337 children, of whom 540 suffered from clinical Pb poisoning. This was in spite of a 1951 prohibition of the use of Pb paint for interior decorating and a 1958 regulation for a warning label on paints containing more than 1% Pb. A house-to-house detection and education campaign succeeded in reducing the annual rate of Pb over-absorption from 10.4 to 7.4 per 1000 children under 4 yr of age. Some measure of success, but not a very notable achievement l Another occasional source of Pb poisoning is the use of old battery cases as domestic fuel (Cited in F.C.T. 1964, 2, 609) and Angle & Mclntire (Am. J. Dis. Child. 1964, 108, 436) report further cases in a family that had been using this type of fuel for several months. This episode first came to light after the youngest member of the family, a 19-month-old boy, had been having convulsions for 8 wk. On the following day his 3-yr-old brother was admitted to hospital with acute encephalopathy and he died on the fourth day of intravenous EDTA therapy. The entire family was then investigated. Two older brothers (aged 4-5 and 7 yr) were found to have high blood Pb concentrations, anaemia and basophilie stippling and symptoms of hyperactivity. The mother, who was 8 months pregnant, had the same haematological changes and also reported fatigue and a colicky abdominal pain unrelated to pregnancy. She received intravenous EDTA therapy for 7 days and a normal baby was delivered 4 wk later. At this time the concentration of Pb in the cord blood was reported to be "negative" or below 0.06 mg/100 ml, no coproporphyrin was found either in the baby's or the mother's urine, maternal haemoglobin had increased and basophilie D

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stippling had disappeared. At the age of 4.5 yr this child was completely normal. This appears to be the first time that treatment for Pb intoxication has been reported during pregnancy and the delivery of a normal baby is evidence of foetal tolerance to this therapy. Earlier work on congenital Pb poisoning shows, however, that there is a definite foetal risk in intra-uterine exposure to high concentrations of Pb in the maternal blood, particularly during the first trimester. A more common source of Pb poisoning in the home is the use of tinned-steel cooking utensils. An investigation reported by Which ? (January 1966) found that 4 out of 9 tinnedsteel frying pans tested contained an alarmingly high proportion of Pb in the tin lining (29-58 ~o) and imparted more than the recommended maximum daily intake (1-2 mg) to bacon, eggs, tomatoes and pancakes cooked therein. Which ? advised housewives to buy this type of frying pan only if they are labelled "pure tinned steel".

Environmental exposure to Pb Another factor to which greater attention may have to be directed is general atmospheric contamination (Cited in F.C.T. 1964, 2, 608). Ludwig et al. (Am. ind. Hyg. Ass. J. 1965, 26, 270) have summarized the results of a joint study conducted between June 1961 and May 1962 by the US Public Health Service, the manufacturers of Pb antiknock additives, the petroleum industry and other appropriate organizations, on the concentrations of Pb in the atmosphere of three cities. The annual average air concentrations of Pb at various locations are reported to range from 1 to 2 pg/m 3 in Cincinnati and from 1 to 3 pg/m 3 in Los Angeles. However, in heavy traffic, air concentrations ranged from 14 to 44 #g Pb/m 3, probably much less than 10~o of this being in organic form. Blood Pb concentrations were determined in 2300 adults and in 1700 of these urinary Pb was also measured. The report states that these concentrations were within the "normal limits", with the exception of 41 individuals with blood concentrations of 0-05 mg/100 g or over and 44 with urine concentrations of 0.07 mg/1 or more, all of whom were subjected to occasional exposure to Pb. It was concluded that the present level of atmospheric pollution by Pb does not constitute a health hazard. At a 1965 symposium on Pb in Washington, spokesmen of the US Public Health Service and the Lead Industries Association were of the same opinion. "At no point during the meeting did anyone show a conclusive clinical tie-in between chronic subtoxic lead exposure and any specific disorder" (Chemical Engineering News 1965, 43 (51), 17). A notalgle absentee from the symposium was Dr. C. Patterson whose strong views founded on Pb geochemistry resulted in the convening of the meeting. A few months earlier, Patterson (Archs envir. Hlth 1965, 11, 344) said that the average resident in the USA is subjected to severe chronic insult due to the industrial use of Pb. In an industrial environment, the average body burden of Pb (200 mg) was estimated to be about 100 times larger than the natural burden in an uncontaminated environment and rates of average Pb absorption about 30 times the natural rates. Patterson (loc. cit.) gave an average concentration of Pb in blood as 0.25 ppm, compared with a threshold limit of 0.8 ppm. The main factor accounting for this increase in body burden was attributed to the use of leaded petrol in motor vehicles (Cited in F.C.T. 1963, 1, 304; ibid 1964, 2, 625). Although faced with the paradox that despite increased environmental contamination by lead since the 1930s levels in man have declined, a view was expressed at the symposium for the need of industry to establish basic environmental health research laboratories. This would greatly assist in pursuing the trend towards "probing the subtle effects of the chemical environment".

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Metabolism and toxic effects of Pb The metabolism of Pb is complex and not fully understood. Following absorption, it is distributed throughout the body and excreted in various inorganic and organic forms. More information is provided by Castellino & Aloj (Br. J. ind. Med. 1964, 21, 308) who studied the kinetics of the distribution and removal of 21°pb from tissues and its excretion in the urine and faeces for 14 days following intravenous injection of 21°pb acetate into rats. In support of previous studies, the highest Pb concentrations were found in red blood cells (96 ~ of the total blood Pb), kidney, liver and bone and in decreasing order in lung, spleen and skeletal muscle. Excretion of 21°Pb in the urine and faeces reached a maximum within 24 hr and after 14 days accounted for just over 50~o of the administered dose. Kinetic measurements of its disappearance from tissues produced three types of curves, an indication of the complexity of Pb metabolism. Approximately 40 ~o of the administered dose was present in bone. 21°Pb disappeared at a slow and constant rate, due to the firm binding of Pb to bone constituents and/or the slow metabolism of bone. In other tissues the initial rapid phases represent the removal of Pb present in ionic form from extracellular and intracellular spaces, and the final slow phase marks the removal of firmly-bound cellular Pb, perhaps as an organic complex. Pb intoxication occasionally causes kidney damage and there is evidence that childhood poisoning with Pb sometimes produces renal failure in later life (Cited in F.C.T. 1964, 2, 273). Kidney damage is readily induced in experimental animals, the epithelial cells of the proximal tubules being mainly affected. Totovi~ (Virchows Arch. path. Anat. Physiol. 1965,339, 15 I) has studied the ultrastructural damage occurring in these cells in rats poisoned with Pb acetate. The changes indicated mild toxic degenerative nephrosis and involved both nucleus and cytoplasm. The nuclei were enlarged and irregular and contained protein- and fat-containing inclusions. The cytoplasm had increased numbers of cytosomes and bodies of complex structure as well as localized deposits of Pb- and calcium-containing crystals, which later formed concentric concretions and sometimes escaped into the tubular lumen. Although these changes are qualitatively similar to those produced by other heavy metals, such as mercury and uranium, they are much less severe probably on account of the deposition in bone of a large part of the absorbed Pb. We are unable to agree with Oliver Wendell Holmes who wrote: "There is no form of lead-poisoning which more rapidly and thoroughly pervades the blood and bones and marrow than that which reaches the young author through mental contact with type-metal."

METHAEMOGLOBINAEMIA: INJECTING NEW LIFE INTO AN OLD PROBLEM Haemoglobin (I) is composed of the pigment haem, conjugated with the protein globin. Haem contains iron in the ferrous state and oxidation to the ferric state results in the formation of methaemoglobin (II) with a loss of oxygen-carrying capacity. In man, II is normally present at 2 ~o of the total content of I in the blood. Three factors are known to produce elevated levels of II (i.e. methaemoglobinaemia): (1) administration of chemical substances; (2) a genetically-determined deficiency in the enzyme system responsible for maintaining II at the normal level; (3) a genetically-determined abnormality in the chemical structure of I. The induction of methaemoglobinaemia by chemical substances is often associated with the formation of round, refractile, eosinophilic particles in the red blood cell, called Heinz

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bodies. The appearance of these inclusions in large numbers is invariably related to a considerable reduction in the number of circulating red blood cells which leads to the development of haemolytic anaemia. Among the many chemical substances known to induce methaemoglobinaemia is the drug, 4,4'-diaminodiphenylsulphone (III). The condition becomes evident at the relatively high doses used in the treatment of the skin disorder dermatitis herpetiformis, but not at the low doses employed in the treatment of leprosy. To investigate whether the drug per se or a metabolite is responsible for these changes, erythrocytes from the blood group O of healthy adult humans were incubated either with normal human plasma to which III had been added, with plasma from patients treated with III or with a rat-liver microsomal preparation containing III (Hjelm & de Verdier, Biochem. Pharmac. 1965, 14, 1119). While II was found absent in the first experiment, levels of 3 and 50 ~o of II were attained in the last two experiments, thus indicating that activity is due to a metabolite, tentatively suggested as a nitroso or hydroxylamine derivative. When the level of II rises above 1.5 g/100 ml of blood (i.e. five times the normal level) in otherwise healthy humans, cyanosis (a blue discolouration of the extremities) is produced without any other clinical manifestations. The presence of this symptomless cyanosis after injection of the local anaesthetic prilocaine (IV) led Hjelm & Holmdahl (Acta anaesth. scand. 1965, 2, 99) to suspect production of II. In a carefully-controlled trial in which IV at doses of 300--1600 mg were administered in single injections to produce local or regional anaesthesia in non-anaemic patients undergoing surgery and in a group of healthy volunteer controls, it was found that peak values of II were reached 6 hr after administration of IV. By 24 hr, lI had disappeared but Heinz bodies persisted for over 40 hr. Administration of ascorbic acid or methylene blue readily prevented or reversed methaemoglobinaemia. Use of other local anaesthetics of differing chemical stucture had no effect on the level of II. Under normal conditions II is converted to I by an efficient reductase system involving nicotinamide-adenine dinucleotide (Cited in F.C.T. 1964, 2, 295) and impairment of this system by foreign compounds will interfere with the oxidation of II to I. Following the addition of sodium nitrite, primaquine or menadione to mammalian erythrocytes in vitro, Tegeris (Toxic. appL Pharmac. 1966, 8, 6) found that whereas sodium nitrite had little effect on the enzyme level the other two compounds produced a pronounced depression. These findings were interpreted to account for the failure of drugs, such as nitrite, to induce Heinz-body formation with resultant haemolysis in vivo, since formation of these inclusions is believed to be dependent upon a certain critical concentration of II (Jandl, Ann. intern. Med. 1963, 58, 702). This critical level is presumably not attained with nitrite, in sharp contrast to the position with the other two drugs. In methaemoglobinaemia a valency change in the haem moiety is far more easily demons° trable than a change in the globin portion. Nevertheless subtle changes in the globin part have been observed by Smith & Gosselin (Toxic. appl. Pharmac. 1966, 8, 159), whose interest was centred on the fact that methaemoglobinaemia protects against the toxic action of cyanide, azide and sulphide. These authors were aware of previous work in vitro, which indicated that these anions form tightly bound complexes with II on a mole per mole basis. When put to the test in vivo, in animals rendered methaemoglobinaemic by nitrite, intravenously-injected cyanide and azide were inactivated by an equimolar amount of II. However, for every 3 moles of sulphide 1 mole of II sufficed and this unexpected result could not be explained in terms of independent factors, such as components of the red cell structure, since the same result was achieved when sulphide was injected into the abdominal

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cavity immediately after an injection of II. That II possesses more binding sites for sulphide than for cyanide is inferred by the finding that cyanmethaemoglobin protects mice against sulphide poisoning, although compared with II the antidotal effectiveness is reduced from a 3: 1 to a 2: I molar ratio. These additional binding sites must therefore be associated with some part of the molecule of I other than the haem iron, which i~ known to be occupied by cyanide. [The subject of methaemoglobinaemia has a close bearing on BIBRA's activities. Several azo colourings are known to produce methaemoglobinaemia and Heinz bodies (Cited in F.C.T. 1963, 1, 102). In a 90-day test on Red 2G in the BIBRA laboratories, considerable attention was paid to this aspect, which represented the only significant finding. Outstanding species differences exist in susceptibility towards chemically-induced methaemoglobinaemia. It is unfortunate that the rat, a widely-used species in toxicity testing, is resistant in this respect. To overcome this difficulty BIBRA is developing a "provocative test" to unmask any latent damage to the red cell of the rat. This test, reference to which was made in the 1965 BIBRA Annual Report (p.25) is based on the superposition of a known methaemoglobinaemia-producing agent on the effects of a test compound.]