Epidemiology for and with a distrustful community.

The epidemiology of diseases associated with chemical waste disposal sites has often been delayed for years after the affected community became aware of the exposure. Frequently, this has resulted in an aroused neighborhood community that is distrustful of those public agencies and officials responsible for protecting their health. It is thus important that positive steps be taken to alleviate the antagonism and to involve the community in an active and constructive role in the epidemiology study. This paper presents a case history of such an incident and highlights some of the lessons learned. The first steps were to involve and inform the community. A citizen and industry advisory committee was formed to participate in the work, publish a newsletter, and conduct regular community meetings. The newsletter and the community meetings were used to identify problems and to explain them; to describe uncertainties of a scientific as well as of political or financial nature; to involve community leaders, including those distrustful; to conduct an epidemiologic census and a neighborhood environmental exposure survey. The end results were a defusing of the antagonism toward authorities, complete acceptance by the community of the merit of the report, a higher quality of report than might otherwise have been possible, and the freedom to move toward alleviating the problems of the disposal site.

INTRACISTERNAL A particles (Bernhard, 1960) have been discovered in both normal and neoplastic mouse tissue (Wivel and Smith, 1971), in a wide variety of transplantable tumours (Kuff et al., 1972) and in early mouse embryos (Biczysko et al., 1973). A previous investigation of MC-induced carcinogenesis in Leaden strain (C57L) mice reported the presence of intracisternal A particles in squamous cell carcinomata but noted their absence from normal epidermis and small papillomata (Kakefuda, Roberts and Suntzeff, 1970). The present preliminary work with BALB/c mice records the presence of A particles in normal epidermis and an increase in their number throughout the early changes in carcinogenesis induced by B(a)P and MC.

MATERIALS AND METHODS
Three-month-old male mice from an inbred BALB/c colony were each housed in a separate box and were isolated in an air conditioned room.
Approximately 24 h before the commencement of treatment, and subsequently when required, the hair from a strip of skin about 1V5 cm wide along the dorsal midline of the mice from the nape of the neck to the tail was removed by electric clippers. The shaved area of the backs of 60 mice was painted with 0 3 ml aliquots of acetone containing either 300 jig B(a)P or 300 ,ug MC on Mondays and Thursdays for a period of 15 weeks. Thirty mice were similarly treated with 0 3 ml ozP/acetone (1/1) and 60 mice were used as untreated or acetone treated controls. One animal from each treatment was killed 3 h after each application and skin from the treated area was removed for electron microscopy. Thirty additional mice were similarly treated with 300 ,tg MC for 20 weeks, after which time carcinomata were removed for electron microscopy.
Pieces measuring 1 mm2 were fixed in 3% gluteraldehyde in 0-2 mol/l phosphate buffer at 4°C, rinsed in buffer and post fixed in 1.33% osmium tetroxide. They were subsequently placed in 2% uranyl acetate, dehydrated in alcohols and embedded in TAAB C resin (TAAB Laboratories, Reading). Sections were cut with a Reichert OMU-3 ultramicrotome, collected on copper grids and stained with lead citrate and uranyl acetate. They were examined on a Philips 301 electron microscope at an accelerating voltage of 80 kV. Examination of untreated and acetone treated skin revealed the presence of occasional intracisternal A particles in the epidermis. They were approximately 65 nm in diameter and the typical doughnut shape associated with A particles. The 2 concentric shells enclosed a comparatively electron lucent centre, the inner shell having a diameter of approximately 40 nm. Particles were observed within cisternae of both rough and smooth endoplasmic reticulum (ER). Characteristically the intracisternal particles form by budding at the ER (Fig.  1). Occasionally small electron-dense granules were present in the epidermal cell nucleoplasm (Fig. 2). These granules varied from ovoid to rectangular in cross section, being approximately 40 nm in length and 25 nm in width. They were predominantly confined to the periphery of the nucleus.
Polycyclic hydrocarbon treatment resulted in initial inflammation accom-panied by hair loss. Sparse hair regeneration followed and papillomata began to appear during the 11th week of treatment. Twelve mice out of 30 had developed squamous carcinoma after 20 weeks' treatment with MC.
Examination of sequential stages in the carcinogenic process with the electron microscope indicated an increase in A particles. In hyperplastic epidermis the particles were individually scattered throughout the cytoplasm and ER (Fig.  3). Large numbers were observed in papillomata, as single individual particles or often in conspicuous groups (Fig. 4). There was no substantial difference in the distribution of A particles between B(a)P and MC induced papillomata. Eight of the 12 carcinomata examined contained a large proportion of cells which were packed with A particles (Fig.  5). A slight increase was observed from the second week of treatment with acP although in none of the animals examined was their incidence as great as in poly-  cyclic induced papillomata and carcinomata. No intercellular particles were observed at any stage during carcinogenesis, even in carcinomata, where large spaces occur between individual cells. After polycyclic treatment there is an increase in the number of nuclei containing electron dense granules in their periphery. This increase appears in parallel with the build up in the number of A particles within the cells. Occasionally papilloma and carcinoma cell nuclei contain a large number of these granules (Fig. 6). There was no increase in these granules after aP treatment.

DISCUSSION
In the present investigation intracisternal A particles have been detected in normal epidermis of BALB/c mice. These particles increase in number throughout the early stages in polycyclic hydrocarbon induced carcinogenesis. Kakefuda et al. (1970) did not detect them in normal or hyperplastic epidermis of Leaden strain (C57L) mice after MC treatment. A number of attempts to demonstrate biological activity associated with A particles have been unsuccessful. However, comparisons of labelling kinetics (Okano et al., 1973) for type A and type C virions in cell culture supported the idea that type A virions are precursors for type C. In the present instance, however, no intercellular particles or intracellular C particles were detected. This supports the current view that in the formation of most type B and all type C particles no true intercellular type A particle is ever involved (Dalton, 1972). Biczysko et al. (1974), investigating the possible morphological sequences of the spontaneous production of an endogenous virus as represented by type A virus particles, described the involvement of round granular structures in the nucleoplasm. Occasionally, similar granules have been detected in the present instance in nuclei of both untreated and polycyclic treated epidermis but have not been observed between the inner leaflets of nuclear membrane. Whether the granular material observed on ultrastructural examination of the nucleus represents a viral precursor and whether the observed increase in granules is related to the increase in the number of A particles induced by polycyclic hydrocarbon treatment are interesting problems. A direct relationship between the proliferation of A particles and neoplastic transformation of BALB/c mouse epidermis appears likely as shortterm non-carcinogenic irritant treatment does not appreciably alter their incidence.