Mining the genome and regulatory networks

A report on the 16th International Conference on Genome Informatics (GIW 2005), Yokohama, Japan, 19-21 December 2005.

XVE report here the results of 4 experiments in which asbestos and other test materials were administered to rats by intrapleural inoculation. These experiments were planned to obtain more information on the carcinogenic effect of asbestos and other materials than could be obtained from our original 2 experiments . Preliminary results of some of the present experiments were given by Wagner, Berry and Timbrell (1970), Wagner (1 970, 1972). In this paper the complete results are given, with emphasis on the light they throw on the aetiology of mesotheliomata, taking into account the oils and waxes present in asbestos, other chemical characteristics and the physical characteristics.

MATERIALS AND METHODS
In all 4 experiments specific pathogen-free (SPF) rats, of the Wistar strain were used. These rats had been bred at the Unit from stocks given to us by Imperial Chemical Industries, Pharmaceutical Division at Alderley Edge, Cheshire in 1964 and1968. The following materials were used: 1. SFA chrysotile.-A super fine sample obtained from a Canadian mine, and produced by water sedimentation separation from grade 7, the most fully milled commercial product. 2. Crocidolite.-Prepared from virgin fibre from a mine in the North West Cape. Both (1) and (2) were from the same samples as used in the earlier experiments .
3. UICC Standard reference samples.-Samples of amosite, anthophyllite, Canadian chrysotile, Rhodesian chrysotile and crocidolite (Timbrell, Gilson and Webster, 1968) prepared following recommendations of l' Union Internationale Contre le Cancer (UICC). 4. Benzene-extracted UICC Standard reference samples-.Samples of (3) which had been repeatedly extracted for 64 hours by hot benzene using a Soxhlet apparatus to remove oils and other benzene-soluble substances. After extraction the benzene was first allowed to evaporate naturally and finally the samples were warmed to 80°C for 24 hours to remove any remaining benzene. After this treatment the samples were tested for the presence of any residual benzene by extracting test portions with cyclohexane and examining the solutions by means of ultraviolet spectrophotometry; no benzene was detected in these solutions. 5. Canadian chrysotiles.-Samples from 8 mines (A, B, . . . H) in Canada. These were the same samples used to prepare the UICC standard reference sample of Canadian chrysotile (Timbrell and Rendall, 1971) but were milled for our purpose more finely than the reference sample.
6. Brucite.-A specimen of brucite, which however also contained chrysotile. This specimen was from Canadian mine H and consisted of long coarse brownish fibres above 50 cm in length. The sample was milled to respirable particle size.
7. Barium sulphate.-Used as a control. This was prepared in the laboratory by the addition of sulphuric acid to barium chloride solution.
8. Saline.-Sterile physiological saline was also used as a control.
9. Ceramic fibre.-A synthetic aluminium silicate fibre. This fibre was prepared for experimental use by grinding in a ceramic ball mill and extracting the respirable fraction by settlement in air. The fibre diameters were between 0 5 and 1 ,um.
10. Fibreglass.-A borosilicate. The nominal diameters of the fibres were between 1-5 and 2-5 ,Lm but in fact only 300% were within this range, the range extending to 7 ,um. The sample was prepared by embedding the fibres in water soluble wax, ejopping in a microtome and washing away the wax. Over 60% of the fibres were longer than 20 ,tm. 11. Glass powder.-A borosilicate all in the respirable range (less than 8 jtm projected area diameter).
12. Aluminium oxide.-A non-fibrous material all in the respirable range (less than 10 ftm projected area diameter).
13. SFA chrysotile (Second sample).-A sample from the same mine and prepared similarly to (1), but taken several years later.
Experiment 1-Varying dose There were 5 doses, 0-5, 1, 2, 4 and 8 mg per rat, of SFA chrysotile and crocidolite. The materials injected were ceramic fibre, fibreglass, glass powder, aluminium oxide, SFA chrysotile and also the second sample of SFA chrysotile. The dose was 20 mg per rat and there were up to 36 rats per treatment (because of a shortage of animals it wAas not possible to allocate 36 to all treatments and in addition inoculation fatalities could not be replaced). Inoculation took place in June and July 1969.
For each experiment animals were allocated at random to treatments. The age of the rats at inoculation was about 6 weeks for Experiments 1, and 3 and 13 weeks for Experiments 2 and 4. In Experiments 1 and 3 there were equal numbers of male and female rats, whilst in Experiment 2 there were 3 times as many females as males, and in Experiment 4 there were twice as many males as females.

Methods
The experimental materials were made up in a suspension of physiological saline with a concentration of 50 mg/ml for Experiments 2, 3 and 4 and for Experiment 1 the concentration was such that the required dose would be present in 0 4 ml of suspension. The rats were anaesthetized with ether and a needle attached to a two-way tap was then introduced into the right axilla at the level of the second nipple. One arm of the two-way tap was attached to a capillary manometer, 1]74 which gave a negative reading when the needle reached the pleural cavity. Details of the method of inoculation were given by Wagner and Berry (1969). Following injection the rats were caged in fours isolated in a special unit. They were fed on a proprietary brand of autoclaved cubes, and water ad libitum. Each rat was allowed to live until it died or appeared to be distressed and a full necropsy examination was carried out, except for a few which had been cannibalized.
The results have been analysed using the model given by Pike (1966) and shown to be valid for experiments of this type . Fuller details are given in in the appendix, and it need only be noted that this method of analysis allows a constant c to be estimated for each of the treatment groups and that this constant, which we will refer to as the " carcinogenicity factor ", serves as a single index summarizing the mesothelioma experience of each group. It combines the information on the proportion of animals which developed a mesothelioma with the times after inoculation at which the mesotheliomata occurred. Also, since the method of estimation eliminates mortality due to other causes, chance variations in natural mortality between different treatment groups do not affect the treatment comparisons, nor do systematic differences in natural mortality between different experiments, such as that resulting from the animals in Experiments 2 and 4 being older than those in Experiments 1 and 3, affect comparisons between experiments.
Where significance levels are quoted they are usually based on the chi-squared approximation to a likelihood-ratio test.

RESULTS
There were 13 rats for which histo-logical examination was not possible, leaving 1112 rats included in the results.
The predominant finding was that a high proportion of most asbestos treated groups developed mesotheliomata and the results will be given mainly in terms of the number of mesotheliomata and the time when they occurred. Sorpe details of the results are given in Tables I-IV. A total of 386 mesotheliomata occurred but the histological features of these tumours will not be described here as there is nothing to add to the features described for the original experiments . Also, in the presentation and analysis of the results, no account has been taken of the sex of the rats. The original experiments show males and females equally likely to develop a mesothelioma, and the present experiments confirm this. Experiment 1 There is a relationship between the number of mesotheliomata and the dose for both SFA chrysotile and crocidolite. This implies that the carcinogenicity is related to dose (d) and we considered this relationship in the form of the carcinogenicity factor being proportional to a power of dose, i.e. c -bdP where b and p are constants. The power p was estimated separately for each dust, giving 073 for chrysotile and 096 for crocidolite. Because of the small number of mesotheliomata, however, these estimates are not very precise and the approximate 95% limits are 0-3-1*3 and 0O2-1-9 for chrysotile and crocidolite respectively. There   are some theoretical grounds for choosing p to be an integer and therefore p was taken as unity. The values of the carcinogenicity factor adjusted to a dose of 20 mg were then 4-10 x 10-9 for chrysotile and 1 70 x 10-9 for crocidolite. Experiments 2, 3 and 4 Comparing first the effects of the separate Canadian samples (Table V), there is considerable variation between Experiments 2 and 3 and this is mainly because of the small number of animals in   There was overall about 30%0 more carcinogenicity in Experiment 2 than in Experiment 3 but the difference is not significant (P > 0.1). Sample C has the lowest carcinogenicity in each experiment and overall is significantly the least carcinogenic (P < 0.05) but apart from this no differences between the samples were detected. These samples have been analysed for certain metals (Holmes, Morgan and Sandalls, 1971;Morgan and Cralley, 1973) and in Table  VI the results of these analyses are shown, together with the carcinogenicitv factor obtained by combining the 2 experiments. The correlation coefficients between the carcinogenicity factor and the different metals are 0-13 for iron, 0-58 for chromium, 0 04 for cobalt, -0-02 for nickel, 0 39 for scandium and 0 04 for manganese. None of these is significant and it is reasonably clear from examination of the chemical properties of sample C that the low carcinogenicity of this sample is not because of a low content of any of these metals.
Turning now to the UICC reference samples (Table VII), there are no significant differences between the normal and benzene-extracted samples, and overall 58 mesotheliomata occurred with the benzene-extracted samples and 56 with the untreated samples. Crocidolite was the most carcinogenic sample with the others in order amosite, anthophyllite, Canadian chrysotile and Rhodesian chrysotile. The difference between the two samples of chrysotile is not significant. Holmes et al. (1971) also carried out chemical analyses on the U7ICC reference samples. There were very large differences between the samples in the amount of the different metals present and it is clear that these bear no relation to the careinogenicitv.
The sample of brucite proved as carcinogenic as the Canadian samples of chrysotile (Table V). Non-asbestos materials which produced the occasional mesothelioma were ceramic fibre, barium sulphate, glass powder and aluminium oxide.
The second sample of SFA chrysotile proved similar in carcinogenic effect to the original sample.

DISCUSSION
The application of the test materials by intrapleural inoculation may be criticized as unrealistic in comparison with human exposure, about which the animal experiments are intended to provide relevant information. Nevertheless, this type of experiment has an important part to play. With an inhalation experiment, which provides a realistic route of entry of the test material, there are 2 factors involved. First, the penetration of dust through the airways and alveoli will differ with different samples of dust (Timbrell, 1965). The second factor is the effect of the dust, given that it has reached the pleura. In inoculation experiments only the second factor is relevant and hence these experiments are simpler to interpret. This makes intrapleural inoculation a more suitable method for the investigation of questions such as whether extraction of the oils alters the carcinogenicity of an asbestos sample. The two types of experiment supplement one another and we will be reporting separately on 2 experiments in which rats were exposed to duist clouds of the UICC reference samples. The varying dose experiment gave results which indicated that the risk of developing a mesothelioma at a given time after injection was proportional to the dose. This form of dose relationship was also found by Pike and Doll (1965) for lung cancer and smoking in man whereas Lee and O'Neill (1971) showed that after repeated applications of benzopyrene to the backs of mice the incidence rate of tumours was proportional to the square of the dose.
The carcinogenicity of the SFA chrysotile sample was similar in Experiments 1 and 2, after adjusting the former to a dose of 20 mg, and in Experiment 4 was lower but not significantly so. In all these 3 experiments the carcinogenicity of the SFA chrysotile was significantly greater than in the earlier experiment  when the estimate of the carcinogenicity factor was 1P68 x 10-9. The crocidolite was also more carcinogenic in Experiment 1 than in the earlier experiment (c 1.16 x 10-9) but not significantly so. These differences could be the result of a change in susceptibility of the rats or of a change in the dust during storage.
The suggestion that natural oils and waxes (Harington, 1962), or contaminating oils from the preparation of the fibre Roe, Walters and Harington, 1966) or from plastic storage bags (Commins and Gibbs, 1969) might contribute to the carcinogenicity of asbestos receives no support from our present experiments, which is in agreement with our original experiments  when removal of the oils from the crocidolite sample resulted in no detectable change in carcinogenicity. Harington and Roe (1965) also advanced the possibility that the presence of trace metals might be relevant to the carcinogenicity of asbestos. In our experiments with the Canadian samples the carcinogenicity was not related to the content of iron, chromium, cobalt, nickel, scandium or manganese. Also, the fact that all the types of asbestos, having very different chemical compositions, produce mesotheliomata makes it unlikely that the carcinogenicity of asbestos could be duie to chemical properties.
Our experiments offer some evidence that the development of mesotheliomata is associated with the presence of fine fibrous material within the pleural cavity. First, UICC Canadian chrysotile is a mixture of batches of material from 8 Canadian mines, and the separate samples used were taken from the same batches (Timbrell and Rendall, 1971). The main difference in the subsequent preparation of the material was that the separate Canadian samples were ground more finely than the composite UICC sample. Comparing Tables V and VII, the carcinogenicities of all the separate Canadian samples were greater than that of the UICC Canadian chrysotile. Also, the samples of SFA chrysotile were from mine D. These were superfine samples and resulted in a very high carcinogenicity. It should be noted that of the Canadian samples the one with the lowest carcinogenicity (C) in both experiments was from a mine in British Columbia whilst the others were from 7 mines in the Quebec area. However, sample C could not be distinguished from the other samples by its size distribution.
A full quantitative analysis of our experimental results will only be possible when techniques are available for complete size characterization of the experimental materials, both before injection and present in the lungs at postmortem. Such techniques to determine the mass and the diameter and length distributions of the particles are being developed. However, even with the characterization methods at present available, a relationship emerges between the observed incidence of mesotheliomata and the physical factors.
A further factor that must be taken into account is the tendency of chrysotile fibres to fragment longitudinally into fine fibrils in lung fluids, the degree of frag-  Wagner,197 To illustrate this electron micrographs materials are presente order of their carcinoge For reasons given by Ti shall consider as " si« those that are less than 0 and also greater than The non-chrvsotile mate sidered first. In the elec for UICC crocidolite amosite (Fig. 4), UIC ( Fig. 5), ceramic fibre ( fibre (Fig. 8) it is evident of " significant" fibres decreasing carcinogenicit-The glass fibre, for instai fibres but the majority ol than 0-5 /tm. On the otl of Carcinogenicity a high proportion by weight of the brucite UICC Reference consists of large fibres, there are also t 3 present a number of very fine long fibrils.
Benzene-It is difficult to compare chrysotile samples cant" fibres present. For example, although the SFA chrysotile (Fig. 1) contains a high proportion by weight of the number of non-fibrous particles, even before injection ced depending on the fibres were in a highly dispersed state. und physiological The enormous number of fibres that types of asbestos, complete fragmentation of chrysotile can wve characteristic produce will be clear from the illustration ions which they that a single fibre may fragmnent into tissue (Timbrell, 1000 fibrils. The fact that this SFA 0o). sample was the most carcinogenic of all relationship, the the materials used corresponds to its of some of the highly dispersed state and its high content d in decreasing of " significant " fibres. nicity ( Fig. 1-8).
The above theory has been examined mbrell (1973), we further using the results of Stanton and gnificant" fibres Wrench (1972). Their experiments were *5 ,um in diameter similar to ours and they used 17 samples, 10 pim in length. including several materials (UICC samples rials will be con-and glass fibres) after partial pulverizatron micrographs tion. They analysed their results by (Fig. 2), UICC discounting submicroscopic fibrils and 1C anthophyllite converting all longer fibres into micro- Fig. 7) and glass fibres of standard size (1 25 x 3 75 ,um) that the number on the assumption that fragmentation of decreases with both glass and asbestos occurred in vivo. ,y of the materials. The numbers of microfibres were then nce, contains long compared with the carcinogenicity of the f these are thicker materials. At first sight their results her hand, whereas seem to conflict with our findings. But,