Quality of Water the Slum Dwellers Use: The Case of a Kenyan Slum

As a result of rapid urbanization in a context of economic constraints, the majority of urban residents in sub-Saharan Africa live in slums often characterized by a lack of basic services such as water and sewerage. Consequently, the urban poor often use inexpensive pit latrines and at the same time may draw domestic water from nearby wells. Overcrowding in slums limits the adequate distance between wells and pit latrines so that micro-organisms migrate from latrines to water sources. Sanitary practices in these overcrowded slums are also poor, leading to contamination of these wells. This study sought to assess sanitary practices of residents of a Kenyan urban slum and fecal contamination of their domestic water sources. This cross-sectional study involved 192 respondents from Langas slum, Kenya. Forty water samples were collected from the water sources used by the respondents for laboratory analysis of coliforms. Of these 40 samples, 31 were from shallow wells, four from deep wells, and five from taps. Multiple-tube fermentation technique was used to enumerate coliform bacteria in water. The study found that most people (91%) in the Langas slum used wells as the main source of domestic water, whereas the rest used tap water. Whereas most people used pit latrines for excreta disposal, a substantial percentage (30%) of children excreted in the open field. The estimated distance between the pit latrines and the wells was generally short with about 40% of the pit latrines being less than 15 m from the wells. The main domestic water sources were found to be highly contaminated with fecal matter. Total coliforms were found in 100% of water samples from shallow wells, while 97% of these samples from shallow wells were positive for thermotolerant coliforms. Three out of the four samples from deep wells were positive for total coliforms, while two of the four samples were positive for thermotolerant coliforms. None of the samples from taps were positive for either total or thermotolerant coliforms. Because the presence of thermotolerant coliforms in water indicates fecal contamination, facilitated by the proximity between the wells and pit latrines, the study suggests that the pit latrines were a major source of contamination of the wells with fecal matter. However, contamination through surface runoff during rains is also plausible as indiscriminate excreta disposal particularly by children was also common. Owing to the fecal contamination, there is a high possibility of the presence of disease pathogens in the water; thus, the water from the wells in Langas may not be suitable for human consumption. To address this problem, treatment of the water at community or household level and intensive behavioral change in sanitary practices are recommended. Efforts should be made to provide regulated tap water to this community and to other slums in sub-Saharan Africa where tap water is not accessible. However, more sampling of different water sources is recommended.
INTRODUCTION
Rapid urban growth in a climate of economic constraints has resulted in the majority of residents in Africa’s large cities, and an increasing proportion of Africans overall, living in overcrowded slums and shantytowns. In these slums and shantytowns, health conditions and livelihood opportunities are poor.1–3 Available evidence indicates that the poor urban residents of Africa exhibit higher morbidity, have poor access to health services, and consequently exhibit higher mortality rates than residents of other areas including rural residents.4–8
The situation in Kenya is similar to other situations in Africa. The proportion of urban population in Kenya nearly doubled between 1980 and 1998, increasing from 16 to 31%.9 Rapid urbanization amid economic degradation in Kenya has resulted in an increased proportion of people living in absolute poverty in the urban areas.10 Therefore, poverty has increasingly become a crucial urban problem in Kenya leading to mushrooming of informal settlements in the urban parts of Kenya where the urban poor find shelter. This has overwhelmed the environmental health resources in urban areas. Because of their illegal status, residents of informal settlements in Kenya do not receive government services such as water, drainage, sewerage, and rubbish collection. Consequently, informal settlements are characterized by poor environmental conditions that predispose their inhabitants to poor health outcomes.4 Evidence shows that children of poor families in urban areas of Kenya exhibit poorer health conditions than their rural counterparts. According to a report by African Population and Health Research Center (APHRC) in 2002,4 infant and child mortality risks are particularly higher in the slums of Nairobi than those observed in other urban areas and in rural Kenya. For instance, the under five mortality was 35% higher among slum residents in Nairobi than among the rural population in Kenya. The report attributes these patterns to poor water and sanitation in these slum settlements.4
An adequate supply of safe drinking water is universally recognized as a basic human need. Yet millions of people in the developing world do not have ready access to an adequate and safe water supply. By 1996, the number of people without access to safe water in urban areas was rising sharply in developing countries as a result of  rapid urbanization, much of which was occurring in peri-urban and slum areas.11 Because the United Nations projects a rapid population growth in urban areas between 2000 and 2030,12 access to safe drinking water and adequate sanitation in urban areas is likely to worsen unless there is a drastic policy change to cater to the needs of the urban poor.
Human excreta and the lack of adequate personal and domestic hygiene have been implicated in the spread of many infectious diseases including cholera, typhoid, hepatitis, polio, cryptosporidiosis, ascariasis, and schistosomiasis. It is estimated that one-third of deaths in developing countries are caused by the consumption of contaminated water and on average as much as one-tenth of each person’s productive time is sacrificed to water-related diseases.13 The World Health Organization estimates that 2.2 million people die annually from diarrhea diseases and that 10% of the population of the developing world are severely infected with intestinal worms related to improper waste and excreta management.14,15 In Kenya, diarrheal diseases are among the major illnesses affecting children of the slum residents. According to the report by APHRC in 2002, prevalence of diarrhea was 32% among children below 5 years of age in the slums, which is double the rate for Nairobi and the national average.4
Where ground water is used as a source of domestic water, use of pit latrines is not recommended because the two are incompatible unless the water table is extremely low and soil characteristics are not likely to contribute to contamination of ground water. Where they coexist, although it is difficult to give a general rule for all soil conditions, the commonly used guideline is that the well should be located in an area higher than and at least 15 m from the pit latrines and should be at least 2 m above the water table. Available evidence shows that increased lateral separation between the source of pollution and groundwater supply reduces the risk of fecal pollution.16 Coexistence of on-site sanitation and use of underground water has in the past been mainly confined to the rural areas where there is adequate land to allow for adequate distance between pit latrines and shallow wells. With the rapid urbanization and rapid expansion of slum settlements in sub-Saharan Africa, on-site sanitation and underground water are used in some urban areas because they are affordable options in the absence of government-supplied services. However, the congestion in the urban slums does not allow for adequate distance between the wells and the pit latrines, which allows micro-organisms to migrate from fecal contents into the underground water sources. Furthermore, poor sanitary practices (for example, disposal of human excreta) in these slum areas lead to contamination of water and consequently water-borne diseases. It is in this context and in the context of high levels of diarrheal diseases in the urban slums in Kenya that this study sought to assess the sanitary practices and the fecal contamination of domestic water sources in an urban slum in Eldoret, Kenya.
STUDY CONTEXT
The study was conducted between January and June 1999 in Langas, an urban slum in Eldoret municipality, Kenya, less than 10 km from Eldoret town. Eldoret town is located in the Rift Valley Province, about 330 km north west of Nairobi. Eldoret, the headquarter of Uasin Gishu District, is one of the fastest growing urban areas in Kenya. Langas falls under high density, low-income areas of the Eldoret municipality. It is divided into four administrative blocks that are further subdivided into about 2,500 plots. Each plot (1/8 of an acre) has between one and 30 households each with an average of six occupants. Settlement in Langas began in 1965 and at the time there were no basic services. Early settlers dug shallow wells for their water needs.17 The water table is high, and this raises the possibility of ground water contamination where on-site sanitation systems are in use.
METHODOLOGY
A cross-sectional study design was used and a sample of 192 households1 was selected through multistage sampling technique as follows: two out of the four administrative blocks were randomly picked and from the two blocks, 192 plots were picked. From each of the selected plots, one household was selected to participate. The 192 households were selected as follows: starting from one corner of each of the blocks and walking across the two blocks in a more or less a diagonal line, about every plot on the diagonal line was selected until the sample of 192 was reached. From each of these plots, one household was selected to represent the plot (this depended mainly on availability of respondents and their willingness to participate), and the first household to be contacted in a selected plot was considered for recruitment into the sample. From the household, a credible respondent was interviewed. A credible respondent was a resident of the selected household who was aged 18 years or above.
For water sampling, 20 samples were taken from water points in each of the two blocks to make a total of 40 samples. This number for water samples was determined by financial and logistical feasibility. In most circumstances, residents of a plot shared one water source and there was roughly one water point per plot. In a few cases, several plots shared a water point. To get the 40 samples, every fourth household2 of the 192 households included in the interview sample was asked their source of water and a water sample was taken from this source so long as it had not already been taken. In the event that a previous fourth household shared the source with the current household, the source for the immediate next household in the study sample was considered. Of the 40 water samples, 31 were from shallow wells (defined as a hand-dug well), four from deep wells (defined as a drilled well) and five from taps (referring to tap water from the municipal council distribution system). Using the above described criteria, only one deep well was selected and purposive sampling was then used to get three other such wells, consequently including all the deep wells that were used by the study sample.
Questionnaires were administered to the 192 selected households to obtain information on the type of toilet facility used, major source of domestic water, method of human waste disposal, whether drinking water was boiled, and the perceptions of possible sources of water contamination in the area. For the method of excreta disposal and water source, the main method and source were considered in instances where there was more than one method or source, respectively. The distance between the pit latrines used by the 192 households and the wells (in cases where they used wells) was estimated. We also observed sanitation practices.
Water samples were collected aseptically with sterile sampling bottles. The samples were transported within 2 hours of collection in a cool box containing ice packs to the Faculty of Health Sciences, Moi University microbiology laboratory for analysis. Fecal contamination of the water was determined through isolation of indicator organisms, total coliforms, and then thermotolerant (fecal) coliforms, through multiple-tube fermentation (MTF) technique. Probability tables (McCrady tables) were used to determine the Most Probable Number (MPN) estimates of the coliform organisms per 100 ml of water. Analysis of data was generally descriptive, involving determination of frequencies. Stata statistical package was used to analyze the data.
RESULTS
Method of Excreta Disposal
The majority of respondents (98%) said that adults used pit latrines, whereas the rest said adults defecated indiscriminately. Similarly, a majority of respondents (70%) said that children used pit latrines, whereas 30% said children used open field/defecated indiscriminately. Most of the pit latrines (95%) in the community were traditional, whereas the rest were ventilated improved pit latrines (VIP latrines). A walk through the community confirmed the report that some people excreted indiscriminately as human excreta was observed strewn all over the compounds.
Source of Domestic Water
Most people (89%) said they used shallow wells as the major source of domestic water, whereas 2% said they used water from deep wells and the rest said they used tap water from the municipal council (Table 1). The shallow wells often had no concrete slab and often the aperture was not covered at all or was poorly covered with a loose lid that was not lockable, whereas the deep wells had a piped system. Those who used deep wells were mainly the more affluent people in the community who often owned the plot in which the well was situated. Tap water was mainly from water kiosks where water was being sold to the slum residents. Respondents who did not use the tap water said that the water from water kiosks was expensive and unaffordable to be used for domestic purposes.3 Problems of unreliability were mentioned as hindering use of tap water from the kiosks as some respondents said that sometimes the kiosk near their house could remain closed for a whole day or more. Some of the respondents reported that the nearest water kiosk was too far from their homes.
TABLE 1Source of domestic water among 192 sampled households in Langas Slum, KenyaSource of domestic waterNumberPercentTap178.9Shallow well17189.1Deep well42.1Total192100
Distance between Pit Latrine and Well
The wells were very close to the pit latrines. In many circumstances (38%), the distance between the wells and the pit latrines was estimated to be less than 15 m (the commonly used guideline is that the distance should be at least 15 m). Most wells (about 59%) were estimated to be at a distance between 15 and 30 m from the pit latrines (Table 2). The distance between pit latrines and wells for the wells from which water samples were taken was similar to that of other wells (Table 3).
TABLE 2Distance between pit latrine and wells for all wells in the study in Langas Slum, KenyaDistanceNumberPercent1–15 m6738.315–30 m10358.930 m and above52.9Total175*100*This total reflects only wells, and thus excludes taps.TABLE 3Distance between pit latrine and well for wells whose water was sampled in Langas Slum, KenyaDistanceNumberPercent1–15 m144015–30 m195430 m and above26Total35*100*This total reflects only wells whose water was sampled, and thus excludes taps.
Boiling of Drinking Water
Despite the short distance estimated between the pit latrines and the wells and the poor sanitary practices like indiscriminate excreta disposal, only 42% of those who reported using wells said they boiled their drinking water, when asked if they did.
Residents’ Perception of Contamination
Respondents pointed out various possible sources of contamination of the water sources in the area. These included children dipping dirty objects into water source (34%) as the main source of contamination, drawing water from the source with dirty containers (27%), domestic animals defecating around water sources (19%), and people washing clothes (5%) at the water source, among others. Interestingly, no one mentioned closeness of the well to the pit latrines as a possible source of contamination.
Water Analysis Results
Forty water samples were analyzed: 31 from shallow wells, four from deep wells, and five from taps.
Wells
Shallow Wells
All the samples (31) taken from shallow wells were positive for total coliforms. The most probable number of total coliforms for most of the samples (71%) was 1,100+ per 100 ml. The minimum number of total coliforms was 63, whereas the maximum was 1,100+ coliforms per 100 ml water.
Only one of the samples had no thermotolerant coliforms at all, whereas most had 1,100+ thermotolerant coliforms per 100 ml.
Deep (Drilled) Wells
Of the four samples taken from the deep wells, three were positive for total coliforms, whereas one was negative. The highest number of total coliforms was 240. For thermotolerant coliforms, two samples were negative. The maximum number of thermotolerant coliforms was 23.
Taps
For the five samples taken from taps (municipal tap water), all of them were negative for total coliforms and consequently thermotolerant coliforms.
DISCUSSION
This paper provides evidence on the extent of contamination of main domestic water sources in the Langas slum and suggests the most probable sources of this contamination. The evidence reveals that the most probable sources of contamination are hardly mentioned among the many sources perceived to contaminate the water sources by the residents of the slum, which impacts how interventions ought to be developed.
The results indicate that the majority of the community members used pit latrines and at the same time used wells as the major source of domestic water. The conditions found in Langas do not fulfill the recommendations given for coexistence of onsite sanitation and use of ground water for domestic purposes, which indicate that there should be an adequate lateral separation between the pit latrine and the well to reduce chances of fecal contamination of the ground water.16 The distance between the wells and the pit latrines was estimated to be generally short with nearly 40% of the pit latrines estimated to be at a distance of less than 15 m from the wells. This raises the risk of contamination of the water sources as coliforms migrate from the pit latrines to the wells.
The presence of indicator organisms (Escherichia coli or thermotolerant coliform bacteria) in water indicates recent contamination of the water source with fecal matter and hence possible presence of intestinal pathogens. According to World Health Organization (WHO) guidelines, E. coli or thermotolerant coliform bacteria should not be detectable in any water intended for drinking.18 The laboratory analysis results of water samples in this study show that fecal matter heavily contaminated the water sources and especially the shallow wells. None of the shallow wells met the WHO requirements for water intended for drinking. The presence of indicator organisms in the water samples collected from the wells indicates the coliforms migrated from fecal matter in the pit latrines through the soil to the water sources, facilitated by the very short distance between the pit latrines and the wells. Poor sanitary practices are also likely sources of pollution of the water sources. Sanitary practices were found to be generally poor from observation and from responses from the respondents. Thirty percent of children excreted indiscriminately and consequently there were a lot of indiscriminately disposed excreta observed. Rains are likely to wash off indiscriminately disposed excreta into shallow wells particularly if the wells are not protected. Therefore, this may have also contributed to the contamination of the generally open shallow wells with fecal matter. Other studies have also attributed contamination of water sources to wet seasons.19 Despite the contamination of water, it was evident that it was not a common practice for the slum dwellers to boil the water, as only 42% of those who used water from wells said they boiled drinking water.
The illegal status of the slum areas in Kenya has hindered the expansion of municipal services to serve them. This has resulted to the poor being denied access to safer drinking water and proper sanitation. The results of this study suggest that water from the tap (which was from the municipal council) was safe for human consumption according to WHO guidelines.18 However, it is important to note that although the presence of thermotolerant coliforms indicates the presence of fecal contamination and potential presence of pathogens, absence of the same does not necessarily mean absence of pathogens, and further investigations would be worthwhile.
Langas is not the only area with the problem of safe drinking water; other urban centers of the developing world have experienced similar problems.19–22 Similar to Langas, in the slums of Nairobi, Kenya, although wells are not a common source of water, slum residents are forced to buy tap water at exorbitant prices from vendors who operate without regulatory mechanisms.23,24 If the Millennium Development Goals of reducing by half the proportion of people without sustainable access to safe drinking water by 2015 and achieving a significant improvement in lives of at least 100 million slum dwellers by 202025 are to be met, there is a dire need for reconsideration of the slum areas in the developing world as far as water supply is concerned, as these slums are home to about 70% of all urban residents in sub-Saharan Africa.26
However, improving the water quality at source alone may not be the ultimate solution because improving water quality at source may not always ensure a reduction in the transmission of water-related diseases. Studies27–30 have shown significant deterioration in water quality between the source and the point of use. Esrey31 concluded that improving water had no health impact if the sanitation was not improved and that improving both water and sanitation together were synergistic in producing larger impacts than either alone. Although the results of this study suggest the need for provision of safer water sources, in this community and in many other slum communities with evident poor sanitary practices, intensive behavior change communication on sanitary practices is also paramount as this has been found effective in the reduction of water-borne diseases elsewhere.32–35
Whereas the findings from this study are worthwhile and act as an eye opener for the situation of quality of water in the rapidly growing informal settlements in the urban areas in Kenya and in the rest of Africa, more sampling of different water sources is highly recommended.
CONCLUSION
It is evident that most of the sources of domestic water in Langas slum are contaminated with fecal matter and do not meet the WHO guidelines for drinking water quality. This poses a health hazard to the residents of the slum as they are at risk of water-borne diseases. The results of this study also suggest that tap water may be safer, but additional sampling is needed. The ideal intervention in the long-run may therefore be provision of adequate piped water to all slum dwellers. However, this may take sometime, and simpler interventions could be put in place in the mean time. Basic sanitary improvement may be worthwhile at the moment. Covering the shallow wells and possibly installing hand pumps or mechanical pumps at the wells could improve the situation. Basic treatment of the water at the community or household level by chemical disinfection using chlorine, filtration using simple household filters, and boiling should also be promoted. These interventions may have a great impact on the health of the slum dwellers as access to safe drinking water and basic sanitation services for populations at risk would result in 200 million/year fewer diarrheal episodes and 2.1 million/year fewer deaths caused by diarrhea.35

water
slums
kenya
urbanization
africa
urban poor
sanitation
sanitary practices
contamination
coliforms
poverty
water quality


The Tobacco Industry and Pesticide Regulations: Case Studies from Tobacco Industry Archives

Tobacco is a heavily pesticide-dependent crop. Because pesticides involve human safety and health issues, they are regulated nationally and internationally; however, little is known about how tobacco companies respond to regulatory pressures regarding pesticides. In this study we analyzed internal tobacco industry documents to describe industry activities aimed at influencing pesticide regulations. We used a case study approach based on examination of approximately 2,000 internal company documents and 3,885 pages of U.S. Environmental Protection Agency documents obtained through Freedom of Information Act requests. The cases involved methoprene, the ethylene bisdithiocarbamates, and phosphine. We show how the tobacco industry successfully altered the outcome in two cases by hiring ex-agency scientists to write reports favorable to industry positions regarding pesticide regulations for national (U.S. Environmental Protection Agency) and international (World Health Organization) regulatory bodies. We also show how the industry worked to forestall tobacco pesticide regulation by attempting to self-regulate in Europe, and how Philip Morris encouraged a pesticide manufacturer to apply for higher tolerance levels in Malaysia and Europe while keeping tobacco industry interest a secret from government regulators. This study suggests that the tobacco industry is able to exert considerable influence over the pesticide regulatory process and that increased scrutiny of this process and protection of the public interest in pesticide regulation may be warranted.
Tobacco is a pesticide-intensive crop. With nearly 27 million pounds of pesticides (including insecticides, herbicides, fungicides, and suckercides) applied to the U.S.-grown crop from 1994 to 1998, it ranks sixth in terms of the amount of pesticides applied per acre [U.S. Government Accounting Office (GAO) 2003]. The tobacco industry regards pesticides as essential to tobacco production, stating that “the crop could not be produced economically without them” (Davis 1989; Philip Morris 1990b). According to industry documents, government-imposed limitations on pesticide use “may present a serious impediment” to the international tobacco trade (Hill 1989).
Internal tobacco industry documents provide a window into the tobacco industry’s activities regarding pesticide regulations. These case studies drawn from industry documents describe the tobacco industry’s responses to pesticide regulatory action. The documents also provide insight into the relationships between the tobacco industry and pesticide regulatory agencies and tensions between business and public health interests.
The Tobacco Industry Documents
Litigation against the tobacco industry has resulted in the release of nearly 7 million previously secret tobacco industry documents (Bero 2003; Malone and Balbach 2000). Scanned PDF versions of original handwritten, typed, or printed documents have been archived at the University of California, San Francisco, library in electronic repositories, searchable using basic keywords (http://legacy.library.ucsf.edu). Between July 2003 and February 2004, we searched the archives using a “snowball” sampling strategy, beginning with broad search terms (“pesticide” and “crop protection agent”) and using retrieved documents to identify more specific search terms (such as names of specific pesticides, people, and regulatory agencies). Table 1 provides examples of keyword searches and the number of documents yielded. This process produced nearly 300,000 documents relating to many different pesticides. The first author reviewed these documents’ index entries and excluded duplicates and documents unrelated to pesticide regulatory issues. The final sample size was approximately 2,000 documents, spanning 1974–2001.
We also filed Freedom of Information Act (FOIA) requests with the U.S. Environmental Protection Agency (EPA) on pesticide issues raised by information in the industry documents, resulting in 3,885 pages of government documents. Finally, we reviewed public health agency reports based on industry documents (Zeltner et al. 2000). We analyzed the industry, government, and public health agency documents by assembling chronologically constructed case studies, a method common to sociology, political science, and anthropology (e.g., analyses of a corporation’s organizational structure, a social movement, or a tribe) (Hill 1993; Yin 1994) (Table 2). The pesticides chosen for inclusion [methoprene, the ethylene bisdithiocarbamates (EBDCs), and phosphine] were those for which sufficient information related to regulatory activities was available in the archives to construct a case study.
Pesticides and Tobacco
Pesticides used on tobacco are also used regularly on food crops. As with food crops, trace amounts of pesticides remain on tobacco leaves after treatment; typically, residue levels decline during the drying and manufacturing process, although additional pesticides may be applied to the finished product (U.S. GAO 2003). Although pesticides increase production of tobacco and food crops, pesticide exposure may harm humans; thus, regulatory agencies such as the U.S. EPA may set limits on the amount of pesticide residue permitted in or on food and tobacco and establish standards for workers handling pesticides. Because tobacco is burned and the smoke inhaled, active and passive smokers are exposed to pyrolyzed pesticide residues (U.S. GAO 2003). The U.S. EPA has concluded that this exposure poses no short-term risk, but little is known about the long-term health effects (U.S. GAO 2003).
Methoprene
In 1974, Philip Morris formed a partnership with the chemical company Zoecon to market a new insecticide (Manzelli 1975). The insecticide’s active ingredient, methoprene, acted as an endocrine disruptor in cigarette beetles and tobacco moths, preventing their larvae from maturing into adult insects (Manzelli 1975). Philip Morris anticipated that methoprene would replace phosphine, a common warehouse fumigant (Philip Morris 1988) and pledged to assist Zoecon in introducing methoprene “in as many countries as we can” (Seligman 1982).
Some countries have regulations that require the establishment of maximum residue limits (MRLs) for pesticides on crops; however, Philip Morris determined that MRLs were not required in all countries, especially for pesticides on nonfood crops such as tobacco (Ryan 1991). Philip Morris asked Zoecon “to not force this issue and submit for MRLs when not required” (Lindahl 1992b). In April 1991 Zoecon alerted Philip Morris’s director of research that the Malaysian pesticide board had recently set an MRL of 1.0 ppm for methoprene on tobacco (Hutney 1991). Zoecon considered 1.0 ppm too low to enable the effective use of methoprene; the level supported by the labeled application rate was 10 ppm (Ryan 1992). Philip Morris requested that Zoecon ask for an even higher MRL of 15 ppm to allow for application errors (Greenberg and Transon 1992; McCuen 1992). Zoecon representatives met with government authorities and requested a change to 15 ppm (Hutney 1991).
A Zoecon representative informed Philip Morris that “in order to avoid surprises of this nature in the future,” he had directed Zoecon’s pharmaceutical group to obtain information from health authorities in other countries regarding the commodities for which methoprene tolerances were assigned (which could include foods such as rice and mushrooms as well as tobacco) (Hutney 1991). Assigning this task to the pharmaceutical group instead of the pesticide group, the Zoecon representative wrote, “will not arouse the curiosity of the health directorates and will allow us to keep our promise to the tobacco industry, namely, that we won’t initiate queries that may cause the health authorities to direct attention to tobacco” (Hutney 1991).
In April 1992, George Lindahl of Zoecon faxed a letter to Bob McCuen, head of Philip Morris’s biochemical research, outlining some of his concerns about Philip Morris’s approach to establishing MRLs for methoprene on tobacco (Lindahl 1992b). In regard to Zoecon’s effort to establish an MRL of 15 ppm in Malaysia, Lindahl explained that
I know we simply argued this case without any data to support our request. In more advanced countries, this tactic will not succeed. … All our data demonstrate the need for a 10 ppm MRL. If a higher value is desired then we will require data from real field operations showing that a worse [sic] case scenario for faulty application will result in a 15 ppm residue, and hence the need for this value. (Lindahl 1992b)
In a fax following this one, Lindahl asked Philip Morris to provide such data; a handwritten comment from a Philip Morris employee who reviewed the fax noted that “data doesn’t [sic] exist” (Lindahl 1992a). Initially, the Malaysian authorities agreed to increase methoprene’s MRL to 10 ppm (Lindahl 1992c); subsequently, it was raised to 15 ppm (Mueller and Ward 1998). Philip Morris continued to advocate (through Zoecon) for MRLs of 15 ppm in Italy and Germany (Greenberg and Transon 1992).
In the meantime, anticipating the creation of a single European market with uniform pesticide regulations, Philip Morris had asked the longtime tobacco industry law firm, Shook, Hardy, and Bacon, to prepare a document with MRL recommendations for possible submission to the European Community (Kemna 1991). Philip Morris first provided a draft of recommended MRLs to the Scientific Working Group of the Confederation of European Community Cigarette Manufacturers (CECCM) (Philip Morris 1991c). At their June 1991 meeting, members of this group (including representatives of Philip Morris, British American Tobacco, R.J. Reynolds, Gallaher, and Rothmans) recommended that the document be rewritten as a voluntary code of practice “to be used pre-emptively … in advance of any EC [European Community] initiative” to impose formal regulations on pesticide residue limits on tobacco (Philip Morris 1991a). A meeting participant reported, “It is hoped that, by implementing this Code, the EC Commission would not any longer see the need to develop a formal EC regulation on pesticide residues in tobacco (products)” (Mueller 1991). Manuel Bourlas, Philip Morris’s director of research and development, was appointed chair of a subgroup of tobacco company representatives who were to assist in preparing the code (Philip Morris 1991a).
This voluntary code underwent numerous revisions throughout 1991 and 1992 (CECCM 1991, 1992a, 1992b, 1992c, 1992d, 1992e; Philip Morris 1991b). Although 236 regulated and unregulated tobacco pesticides were in use at the time (Mitchell 1991b), the voluntary code proposed MRLs for only 25–27 pesticides [including chlordane, dichlorodiphenyltrichloroethane (DDT), lindane, dithiocarbamates, methoprene, and maleic hydrazide]. According to British American Tobacco’s Terry Mitchell, “many of the substances in the list are no longer recommended for tobacco production” (e.g., DDT) (Mitchell 1991a). Moreover, this list did not impose “any constraint automatically on non-specified substances” (Mitchell 1991a). Mitchell noted that this lack of limits was “highly desirable” (Mitchell 1991a).
In December 1992, Walter Russell, a legal assistant, reported that the code “has undergone two more revisions (by SHB) [Shook, Hardy, and Bacon] and it [is] currently watered down, but still causing much agitation” (Philip Morris 1992). Russell pointed out that the code set MRLs that Philip Morris “might have trouble complying with” if they were to become international standards (Philip Morris 1992). In addition, “failure to comply with tolerances written by the tobacco industry which might come up during litigation would put the tobacco industry at great disadvantage” (Philip Morris 1992). He indicated that Philip Morris had decided to withdraw its support from the voluntary code (Philip Morris 1992). In 1993, the tobacco companies suspended work on the document due to “principle disagreements both within and between participating companies” (R.J. Reynolds 1993). Throughout the 1990s, the tobacco industry continued to anticipate European Union harmonization of tobacco pesticide MRLs (Philip Morris 1995); as of April 2004, the European Union had established community-level MRLS for 150 pesticides, but none specifically applied to tobacco (European Union 2004).
EBDC Fungicides
In 1987, the U.S. EPA initiated a review of EBDC fungicides, prompted by the agency’s determination that a breakdown product of EBDCs, ethylene thiourea (ETU), was a probable human carcinogen (U.S. EPA 1987). Anticipating the U.S. EPA’s cancellation of many EBDC uses, U.S. manufacturers voluntarily withdrew EBDC registrations for all but 13 food crops in 1989, including wheat and corn (U.S. EPA 1989). At least one company continued to hold registrations for EBDCs on tobacco, but only for seed bed use, not plants (Arce 1989).
In internal documents, the tobacco industry expressed concern that the U.S. EPA’s action could result in the “imposition of potentially crippling product residue tolerances” in Europe [Centre de Coopération pour les Recherches Scientifiques Relatives au Tabac (CORESTA) 1989b; Mitchell 1990]. EBDCs were regarded as vital to control blue mold outbreaks in Europe (Philip Morris 1990a). In October 1989, members of CORESTA, an international tobacco research organization with members drawn largely from the tobacco industry, established a subcommittee to “provide regulatory agencies with a sound basis for the development of tobacco agro-chemical regulations” (CORESTA 1989a, 1989b).
As discussed in a larger World Health Organization (WHO) report on tobacco industry influence at that agency, the subcommittee hired a consultant, Gaston Vettorazzi, to provide advice on influencing regulation (CORESTA 1990b; Zeltner et al. 2000). Vettorazzi was a former WHO toxicologist and former technical secretary of the Joint Food and Agriculture Organization/WHO Meeting on Pesticide Residues (JMPR), an international meeting of scientists whose decisions often formed the basis of international law (Zeltner et al. 2000). Selected partly for his “old boys’ contacts” (Reif 1991b), Vettorazzi’s initial duties were to provide a review and analysis of toxicologic data on EBDCs and ETU (CORESTA 1990a).
Some CORESTA members were concerned that Vettorazzi’s review might conclude that EBDCs were unsafe (Beuchat 1990). However, according to one member’s notes, at his first meeting with the subcommittee in April 1990, Vettorazzi stated that “someone has to lay the red carpet for [me], otherwise [I] can spoil more than help” (Reif 1990).
Vettorazzi’s initial review concluded that ETU was neither carcinogenic nor genotoxic (Vettorazzi 1991a). Some of the tobacco industry scientists commented that this statement was “too strong in light of the NTP feeding studies”—a reference to the U.S. National Toxicology Program’s conclusion that animal studies showed clear evidence of ETU’s carcinogenicity (Reif 1991a). Vettorazzi subsequently revised his conclusions, stating that ETU’s “toxicity, including carcinogenicity, can be explained by the known mechanisms of action characteristic of thyroid-function inhibiting agents” (Vettorazzi 1991b). Thus, he stated, a threshold could be set below which ETU did not cause thyroid tumors (Vettorazzi 1991b).
CORESTA authorized the distribution of Vettorazzi’s revised report to his former colleagues at WHO, once all references to tobacco and CORESTA were removed (CORESTA 1992). WHO’s JMPR was scheduled to review EBDCs/ETU in 1993; if this review were favorable, the tobacco industry would be assured continued access to EBDCs in Europe (Zeltner et al. 2000).
With CORESTA funding ($100,000 a year) and approval, Vettorazzi offered to assist J. Herrman, of the JMPR WHO Secretariat, with JMPR toxicologic reviews, without disclosing his tobacco industry ties (Herrman 1991; Vettorazzi 1991c, 1992a). Vettorazzi wrote and reviewed several working papers on compounds to be discussed at the 1992 JMPR, including the EBDC thiram (Herrman 1992; Vettorazzi 1992b). One outcome of that meeting was the reestablishment, at a higher level, of the previously cancelled Acceptable Daily Intake (ADI) for thiram (Vettorazzi 1992b).
Vettorazzi continued his work with WHO in 1993, supplying his CORESTA-funded reviews to the adviser responsible for drafting the working paper that would form the basis of the September JMPR on EBDCs/ETU without revealing their sponsor (Zeltner et al. 2000). Vettorazzi also attended the September meeting as an invited “temporary adviser” (Zeltner et al. 2000). The meeting’s outcome reflected Vettorazzi’s conclusions. In contrast to the U.S. EPA, JMPR determined that ETU was not genotoxic, and thus raised the ADI level from 0.002 to 0.004 mg/kg body weight (Black 1993). CORESTA considered this “a very positive result for the industry,” since it “clearly indicates that the ‘carcinogenicity’ of [ETU] is not really a burning issue any longer” (CORESTA 1994; Mueller 1993). JMPR’s safety standard became part of international trade law, preserving tobacco industry access to EBDCs (Zeltner et al. 2000). Soon after the JMPR meeting, CORESTA extended Vettorazzi’s contract for 18 months, listing one of his duties as providing “information about the activities of pesticide action groups” (CORESTA 1993). He was to be paid another $100,000 (CORESTA 1993). Vettorazzi continued working for CORESTA until at least 2001, when the organization paid him $30,000 to monitor international activities related to tobacco pesticide residues and registrations (CORESTA 2001).
Phosphine
Phosphine is a fumigant used on stored commodities, including nuts, seeds, grains, coffee, tobacco, and finished cigarettes to kill insects. Because of the risks it poses, applicators are advised to wear respirators and protective clothing, and warehouses must be sealed to prevent leaks that contribute to air pollution and endanger nearby residents (U.S. EPA 1998b). By the early 1990s, several case reports had been published noting sometimes fatal phosphine poisoning among workers and community members (Garry et al. 1989, 1993; Heyndrickx et al. 1976; Schoonbroodt et al. 1992; Wilson et al. 1980).
In December 1998, the U.S. EPA proposed a series of 15 risk mitigation measures (RMMs) for phosphine. The U.S. EPA’s primary concern was the risk that phosphine posed to applicators and community residents (U.S. EPA 1998b). Thus, the RMMs included a threshold limit value of 0.03 ppm of phosphine during fumigation (reduced from the existing 0.3-ppm standard), the establishment of a 500-foot buffer zone around all fumigated structures, and prior notification of all residents living within 750 feet of a fumigated structure (U.S. EPA 1998a).
The Tobacco Association of the United States, in a letter to the U.S. EPA, stated that the economic burdens imposed by the RMMs would “make it virtually impossible for our industry to continue to fumigate stored tobacco” (Ward 1999). The Tobacco Association, R.J. Reynolds, Philip Morris, and > 150 other organizations with a stake in the continued use of phosphine formed a lobbying group, the Commodity Industry Coalition for Phosphine Fumigation (Harrell 1999).
R.J. Reynolds, represented primarily by toxicologist Joel Seckar, took an active role in the Commodity Industry Coalition (Seckar 1999c). The company calculated that complying with the U.S. EPA’s buffer zone requirement would cost approximately $50 million in new land and warehouse purchases (R.J. Reynolds 1999a). Increasing the time required to aerate warehouses before employee reentry to comply with the worker exposure limit of 0.03 ppm would increase costs, as would the possibility of liability suits brought by nearby residents notified of phosphine use (Degesch America 1998; R.J. Reynolds 1999d).
Coalition members lobbied Congress, released media statements, worked closely with the U.S. Department of Agriculture, and attended U.S. EPA-sponsored stakeholder meetings (Goldman 1998; Lyon 1999; R.J. Reynolds 1999b, 1999c). Their message was that the proposed RMMs were overly conservative, based on “anecdotal information and hypothetical risk” rather than on “sound science” (Lyon 1999; Ong and Glantz 2001). To challenge the scientific basis of the U.S. EPA’s proposals, the coalition decided to hire an expert whose research would support existing standards (Seckar 1999h). They chose Sciences International, a consulting firm specializing in health and environmental risk assessment. It was headed by Elizabeth Anderson, a former director of the Carcinogen Assessment Group and the Office of Health and Environmental Assessment at the U.S. EPA (Sciences International 2005). She was also an experienced expert defense witness, having served in that capacity in a number of environmental lawsuits brought against corporations (Anderson 1999c).
To support the Commodity Industry Coalition’s assertion that the proposed exposure level of 0.03 ppm was too conservative, Sciences International focused on the inter-species uncertainty factor. The U.S. EPA had first determined from a published subchronic toxicity study of rats that there were no observed effects attributable to inhaled phosphine at 3 ppm (Seckar 1999a). To extrapolate to humans, the U.S. EPA had then used two 10-fold uncertainty factors, one for intraspecies variability and one for interspecies variability, to arrive at a maximum exposure level of 0.03 ppm (Sciences International 1999c). Documents indicate that Sciences International’s strategy was to convince the U.S. EPA that the interspecies uncertainty factor was unnecessary, showing that because a number of animal species reacted in the same manner to phosphine, humans were similar enough that the interspecies uncertainty factor could be removed (Seckar 1999a, 1999b). This would leave only the intraspecies factor of 10, which would result in a maximum exposure level for humans of 0.3 ppm, the existing standard.
In April 1999, the U.S. EPA representatives met with a small group of Commodity Industry Coalition members, including R.J. Reynolds’s Seckar and Sciences International’s Anderson (Seckar 1999a). Anderson questioned the U.S. EPA’s interspecies uncertainty factor, citing several animal studies and an epidemiologic study to suggest that the U.S. EPA’s calculations were too conservative (Seckar 1999a). In an e-mail, Seckar noted that Anderson’s presentation was very effective, as evidenced by the fact that U.S. EPA representatives were now informing coalition members that the 0.03 ppm standard “was not ‘set in stone,’” a direct contradiction of earlier statements to the U.S. Department of Agriculture (Bair 1999; Seckar 1999d). (Despite Freedom of Information Act requests, we were unable to obtain U.S. EPA documents related to its meetings with the coalition.)
Soon after, Sciences International asked the Commodity Industry Coalition for additional funding to turn its phosphine report into a peer-reviewed journal article (Turim 1999). In a memo to Seckar, Anderson (1999b) explained that
My experience is that consultant reports funded by those being regulated, and written expressly for the EPA, are easily and frequently ignored or dismissed by the Agency, no matter how scholarly. However, a paper or article that is peer-reviewed and published, or in the peer review process for publication, in an accepted scientific journal can neither be ignored nor dismissed.
Anderson suggested that since she was editor-in-chief of Risk Analysis, “perhaps the peer review process could be expedited if we decide that it is the journal of choice” (Anderson 1999b). R.J. Reynolds, Brown and Williamson, and several other tobacco companies agreed to fund most of the cost of this work (Seckar 1999e). The paper was published in Risk Analysis in 2004, with the acknowledgment that “This work was supported by the Phosphine/Metal Phosphide Coalition, consisting of the producers and users of phosphine and metal phosphides for the control of insects in stored commodities” (Pepelko et al. 2004).
Coalition members also pursued other strategies. At a meeting with U.S. EPA representatives in March 1999, the Commodity Industry Coalition proposed that the U.S. EPA participate in a series of small, coalition-sponsored focus groups to “educate [EPA] on the issues involved with … fumigations” (Seckar 1999g). One such group met in May 1999, when tobacco companies demonstrated a tobacco warehouse fumigation (Ward and Cowan 1999). The following month, several companies conducted additional emissions tests to show that the proposed 500-foot buffer was unnecessary (Bridges 1995). However, an e-mail message from a Philip Morris employee indicated that Philip Morris’s test coordinator had “some reservations regarding the quality of the test design/data generation” and that he himself believed that “the test plan and methods will provide, literally, no information, so it won’t hurt us to do it” (Bridges 1995).
In June 1999, Sciences International submitted a first draft of its phosphine toxicity review to some coalition members (Sciences International 1999a). A reviewer from the coalition’s lobbying firm pointed out that the animal studies cited did little to support the idea that the interspecies uncertainty factor should be eliminated “since most [of the animals] appear to be rat or mouse strains with similar breathing characteristics” (Wilkinson 1999). Instead, the studies cited by Sciences International seemed to support the idea that phosphine called for a conservative standard, as they indicated that “phosphine is a very toxic material to most species tested” (Wilkinson 1999). Another reviewer noted that the uncertain and tentative tone of the report “will trigger concerns by EPA and they will say ‘if [an] expert in the field states that there remains great uncertainty, maybe we are on solid ground by being very conservative’” (Barolo 1999a). Sciences International staff revised the report, removing tentative statements and asserting that their work to date supported reducing the interspecies uncertainty factor to 1 (effectively eliminating it), thus preserving the existing exposure standard of 0.3 ppm (Sciences International 1999b). They submitted this revised interim report to the U.S. EPA in July 1999 (Sciences International 1999b). At a Commodity Industry Coalition meeting that same month, coalition consultant Dan Barolo, former director of the U.S. EPA’s Office of Pesticide Programs (OPP), reportedly urged members to speed their efforts because
phosphine is quite hazardous when used improperly. The more the Coalition slows the process, the greater the chance for an accident with possible fatalities, which would send EPA back into conservative mode and make it far more difficult for them to publish reasonable RMMs. (Seckar 1999f)
In August, John Whalan, a toxicologist at the U.S. EPA’s Health Effects Division, summarized in a memo his analysis of Sciences International’s interim report (Whalan 1999). He noted that
there is no precedent for using an [interspecies uncertainty factor] of 1 when establishing … an inhalation regulatory value in the Health Effects Division. The only time an interspecies [uncertainty factor] is not applicable is when human data are used. The available data do not support deviating from Agency policy, and the Coalition did not provide any new data. (Whalan 1999)
He also pointed out that Sciences International’s review of animal studies, intended to show that phosphine toxicity was relatively constant across species, was largely “irrelevant” because it did not include a comparison of toxicity for a small versus large mammal.
In September 1999, phosphine registrants and several coalition members again met with U.S. EPA officials to discuss alternative RMMs proposed by the coalition (Seckar 1999i). Instead of a 500-foot buffer and a 750-foot neighbor notification requirement, the coalition recommended a “site management plan” that required companies to develop emergency preparedness measures. The U.S. EPA asked the Commodity Industry Coalition to reword its proposals to specify how and when workers and bystanders would be informed of danger (Seckar 1999i). On the exposure limit for workers, the U.S. EPA now proposed a 0.1-ppm standard (reflecting a reduction from 10 to 3 in the interspecies uncertainty factor) based upon Sciences International’s interim report (despite the weaknesses noted by Whalan) (Seckar 1999i). (The U.S. EPA failed to provide memos or notes regarding this decision.)
In several fall 1999 memos to Seckar, Sciences International staff explained that they thought it would be difficult to convince the U.S. EPA to drop the interspecies uncertainty factor without human exposure studies (Anderson 1999a; Gray 1999). Commodity Industry Coalition members expressed reluctance to commit to human studies without confirmation that this would convince the U.S. EPA to “give up” the uncertainty factor (Barolo 1999b). Barolo commented to Seckar, “I do not believe it will be easy for OPP to abandon both safety factors. There are too many unknowns from children to endocrine to reliability of studies to absence of dog/monkey study. … Some day they are going to figure out there is a 0.1 ppm standard in other countries and the door will close” (Barolo 1999c).
Although Sciences International had not yet submitted to the U.S. EPA its full report on phosphine, in December 1999, the U.S. EPA made its final decision (Sharp 1999). (This decision was published in the Federal Register in February 2001 [U.S. EPA 2001]). The U.S. EPA now mandated a “fumigation management plan” like that proposed by the Commodity Industry Coalition (U.S. EPA 2000). The agency also eliminated the inter-species safety factor and left the old 0.3-ppm standard in place, on condition that phosphine registrants conduct additional research if Sciences International’s review was found to be inadequate (U.S. EPA 2000). A coalition member noted that “it is important to point out that this additional work will take years and that the current 0.3 ppm threshold will stay in place during that time” (Sharp 1999). R.J. Reynolds credited its leadership on the scientific issues with saving the company “many millions of dollars” (R.J. Reynolds 2000).
Conclusion
Although others have charged that agencies responsible for protecting human health and the environment are unduly influenced by the industries they regulate (Abraham 2002; Huff 2002), it is rare to be able to study this process from the perspective of the regulated industry. This study provides documentation of the behind-the-scenes activities of an industry as it attempts to influence the regulatory process on matters that have a direct bearing on public health.
Our analysis has limitations. Given the sheer volume and limited indexing of the documents, it is impossible to ensure that we located all potentially relevant documents. Some may have been destroyed or concealed by the tobacco companies (Liberman 2002); others may have never been obtained in the legal discovery process. In addition, we had no access to pesticide company documents, except those in the tobacco documents archives. Finally, despite properly filed Freedom of Information Act requests, we were unable to obtain from the U.S. EPA documentation of its meetings with the industry’s Commodity Industry Coalition. All minutes of meetings with stakeholders should be part of the public record.
Despite these limitations, the case studies discussed here provide insight into tactics that the tobacco industry applies to a regulatory agency when trying to influence the outcome of a decision. These tactics go significantly beyond the usual approaches—such as participation in public comment periods and public meetings—to influence scientific and regulatory decision making. Tobacco industry tactics described in these cases include:
Encouraging a chemical company (Zoecon) to advocate for high MRLs without any supporting data and directing that same company to gather information about international regulatory efforts on methoprene in a manner designed to hide the interest of the tobacco industry in this chemical;
Attempting to forestall regulatory efforts on tobacco pesticides in the European Community by creating voluntary industry MRLs for a subset of chemicals;
Hiring an ex-WHO scientist to participate (without disclosing his funding source) in the WHO regulatory effort on EBDCs;
Hiring several ex-U.S. EPA scientists to influence the U.S. EPA’s regulatory decision making on phosphine;
Hiring scientific consultants with instructions to marshal data to support the tobacco industry’s a priori arguments and funding consultants to publish a report supporting these arguments in a journal over which the consultants had influence;
Staging fumigations for the U.S. EPA with the knowledge that the methodology was flawed and the results would show no emissions problem.
Yet, as the case of European MRLs showed, the tobacco industry does not always work together effectively to influence regulations. Tobacco companies may disagree about regulatory strategies or conclude that inaction is preferable to action that might have unintended consequences. Moreover, the fact that even voluntary, industry-friendly pesticide guidelines posed significant problems for Philip Morris underscores tobacco industry motivation for resisting or influencing more stringent, government-imposed regulations.
This study also raises questions about industry influence over regulatory agencies. In the case of WHO deliberations on EBDCs, the tobacco industry coordinated covert actions, hiding the financial ties and involvement of CORESTA. Rigorous disclosure requirements and oversight might have allowed the WHO’s agencies to judge more accurately the potential for bias related to conflicts of interest. In the case of the U.S. EPA’s review of phosphine, a regulatory agency appears to have been quite willing to cooperate with the industry and its consultants. This is a reminder of why regulatory processes were designed to be transparent and open to the public, and why “closed-door” meetings between regulators and industry have been ruled illegal (Federal Advisory Committee Act 1972; Registration Standards 2004; Special Review Procedures 2002).
Protection of the public interest hinges on an open process and regulatory agencies’ willingness to stand up to pressure from regulated industries. When these are in doubt, public confidence in the fairness and efficacy of regulations may be unwarranted. The resource disparities between powerful industries and public health organizations may also make it difficult to ensure that the public interest is fairly represented, particularly when discussions occur behind closed doors, as apparently occurred at the U.S. EPA. Increased public and media scrutiny of these processes could help ensure that public health considerations are weighed at least as heavily as commercial ones.
Finally, given the deadly epidemic of tobacco-caused disease, which kills an estimated 5 million people annually worldwide (WHO 2004), is it in the public interest for regulatory agencies today to continue facilitating standards that make it easier and less costly to grow, transport, store, and manufacture tobacco products?

tobacco industry
pesticide regulation
environmental protection agency
methoprene
ethylene bisdithiocarbamates
phosphine
world health organization


Purinergic signalling in the subretinal space: a role in the communication between the retina and the RPE

The retinal pigment epithelium (RPE) is separated from the photoreceptor outer segments by the subretinal space. While the actual volume of this space is minimal, the communication that occurs across this microenvironment is important to the visual process, and accumulating evidence suggests the purines ATP and adenosine contribute to this communication. P1 and P2 receptors are localized to membranes on both the photoreceptor outer segments and on the apical membrane of the RPE which border subretinal space. ATP is released across the apical membrane of the RPE into this space in response to various triggers including glutamate and chemical ischemia. This ATP is dephosphorylated into adenosine by a series of ectoenzymes on the RPE apical membrane. Regulation of release and ectoenzyme activity in response to light-sensitive signals can alter the balance of purines in subretinal space, and thus coordinate communication across subretinal space with the visual process.
Introduction
The retinal pigment epithelium (RPE) lies between the outer segments of the photoreceptors and the choroidal blood supply (Fig. 1). The RPE combines the functions of epithelial and glial cells, providing a barrier while also supporting the neural photoreceptors and modulating their function. Tight communication between photoreceptors and the RPE is critical to coordinate the multiple levels of interaction, and the purinergic contribution to this communication is becoming apparent. The relevance of this purinergic input is emphasized by the many functional effects of P1 and P2 receptor stimulation and by the multiple mechanisms in place to regulate subretinal levels of purine agonists. As the dynamics of ATP release and extracellular conversion into adenosine will modify agonist availability, the modulation of these processes can exert a temporal control on purinergic signaling. The following review will outline the main interactions between the RPE and photoreceptors, describe the effects of stimulating purinergic receptors on both sides of subretinal space, and summarize how levels of ATP, ADP, and adenosine are manipulated in this microenvironment.
Fig. 1Schematic illustration of the key components of purinergic signaling in the subretinal microenvironment. Stimulation of P2 receptors on the RPE can enhance transepithelial fluid absorption while P1 receptors can modulate phagocytosis. ATP released through CFTR and other Cl− channels can stimulate P2 receptors or be converted to ADP, AMP, and adenosine (Ado) by a series of ectonucleotidases present on the apical membrane of the RPE. By controlling the balance of extracellular purines available to stimulate these receptors these mechanisms can control levels of endogenous purines available to activate the receptors. While theoretically possible, it remains to be determined whether these subretinal purines can actually stimulate photoreceptors
Purines and subretinal space
RPE-photoreceptor interactions across the subretinal space
The outer segments of the rods and cones are responsible for the initial stages of vision, converting photon energy into a series of enzymatic reactions that close the light-sensitive channels on the photoreceptor plasma membrane, hyperpolarize the cells, and reduce the release of glutamate from the synaptic terminals [1, 2]. Efficient photoreceptor function depends upon both short-term and long-term support from the RPE. The critical nature of these interactions is evident from the rapid degeneration of photoreceptors in the absence of a healthy RPE layer and by the RPE localization of defective gene product in some forms of hereditary photoreceptor degeneration [3].
The apical membrane of the RPE is separated from the plasma membrane of the outer segments by an extracellular space of only 10–20 nm [4]. Although small, this subretinal space contains a highly structured matrix which ensheathes the outer segments and extends to the RPE [5, 6]. The presence of enzymes within this interphotoreceptor matrix emphasizes that this extracellular space itself is functionally active [7, 8]. This intimate anatomical relationship between photoreceptors and the RPE reflects multiple functional interactions. For example, the RPE accepts, recycles, and exports central components of the phototransduction process [9]. The outer segments are continuously resynthesized, and the phagocytosis, degradation, and processing of shed outer segment tips by the RPE cells is central to this renewal [10]. The ion channels and transporters on the apical membrane of the RPE help regulate the ionic composition of the subretinal space [11]. As extracellular levels of ions can modify the ionic driving forces across the photoreceptor plasma membrane, these RPE transporters can influence the state of neural activity. The transport of fluid and ions from the apical membrane to basolateral membrane of the RPE is also one of the main forces keeping the retina attached [12].
The control of photoreceptor activity by light gives a rapid temporal dependence to some interactions between the photoreceptors and the RPE. The volume of subretinal space increases in response to light [13], linking small changes in the ionic composition of the subretinal space with activity of the RPE transport mechanisms which maintain this volume [14, 15]. Other processes are controlled on a diurnal cycle. The shed tips of the outer segments are phagocytosed by the RPE soon after the onset of light [16, 17]. These processes can both be modulated by purine levels in subretinal space, indicating purinergic regulation is important over multiple time scales.
Purinergic receptors on photoreceptors
A2 adenosine receptors were localized to both the inner and outer segments of photoreceptor outer segments over a decade ago by Blazynski and colleagues [18], with more recent reports emphasizing their functional role. A2 agonists inhibit the L-type Ca2+ channel on rod outer segments [19] and can inhibit the synaptic release of glutamate from rods, suggesting changes in adenosine levels in subretinal space could modulate light sensitivity [20]. The A2 agonist DPMA and the adenosine deaminase inhibitor EHNA reduce mRNA for opsin in rods, suggesting that endogenous levels of adenosine can downregulate opsin message at night [21]. EHNA and the A2A receptor agonist CGS21680 also increase the survival of chick embryonic photoreceptors in culture [22], indicating a long-term neuroprotective role for adenosine.
P2 receptors are also present in the photoreceptors. mRNA for the P2X2 receptor is expressed in the photoreceptor cell bodies, with immunohistochemical localization of the protein to outer segments [23]. In situ hybridization indicates the photoreceptor layer has the highest level of P2Y2 receptor of any region in the rabbit retina, although staining was not pronounced in monkey [24]. P2X7 receptors have recently been localized to photoreceptor synaptic terminals, with evidence for ecto-ATPase activity in the synapse, and functional evidence suggesting ATP augments transmission of the light response by rods [25]. It was suggested that ATP might be co-released from photoreceptors with glutamate, although this remains to be tested directly.
Purinergic receptors on the RPE
Stimulation of P1 receptors can have a considerable impact on RPE cells. A2 receptors have been recognized on cultured and fresh RPE cells for some time [26, 27], with in situ hybridization confirming the presence of A2A receptors in rat RPE [28]. Stimulation of A2 receptors reduces the rate of rod outer segment phagocytosis by RPE cells [29], while application of adenosine to the apical membrane of chick RPE cells increases the basolateral Cl- conductance, the transepithelial potential, and the c-wave, and decreases the hyperpolarization of the basal membrane in response to light [30]. Although adenosine alone does not increase intracellular Ca2+ levels [31], adenosine acts synergistically with ATP to elevate Ca2+ levels in human RPE cells by stimulating both A1 and A2A receptors [32, 33]. Stimulation of A1 receptors with high doses of NECA increases the active transport of fluorescein across the RPE, while activation of A2A receptors decreases this transport, and by extension, transport of the ions that underlie fluid movement [34]. Stimulation of A1 and A2A receptors produces analogous increases and decreases, respectively, in the absorption of subretinal fluid blebs. This is consistent with the negative coupling of the A1 receptor and the positive coupling of the A2 receptors to adenylate cyclase, as increasing cAMP inhibits the transport of fluid across the RPE towards the choroid [35–37]. The agonist 2-Cl adenosine reverses the deficit in phosphoinositide metabolism found in diabetic RPE cells [38], suggesting effects on metabolism in addition to transport and phagocytosis.
Multiple P2 receptors have been localized to the RPE. The P2Y2 receptor was initially characterized in cultured human RPE [31], with subsequent reports localizing transcript for P2Y1, P2Y2, P2Y4, and P2Y6 in the rat RPE/choroid [39] and for P2Y1 and P2Y12 receptors in ARPE-19 cells [40], and functionally identifying a P2X receptor in rat RPE cells [41]. ATP, ADP, and UTP induce numerous effects on RPE physiology [32, 33, 42, 43]. While these effects likely involve multiple different receptor types, the contributions of the P2Y2 receptor have been explored in most detail to date. The P2Y2 receptor has been specifically localized to the apical membrane of fresh bovine RPE cells, and addition of ATP to this membrane transiently elevates Ca2+, activates a basolateral Cl- conductance, inhibits an apical K+ conductance, and increases the apical to basolateral flow of fluid [43]. This increased absorption of fluid from the subretinal space suggests P2Y2 receptor stimulation could reduce the excess fluid that accumulates in retinal edemas, and several reports have supported this theory. ATP, UTP, and the P2Y2 receptor agonist INS37217 decrease the size of subretinal fluid blebs when injected into subretinal space of rats [44]. In both normal and rds +/- mice with experimentally induced detachment, INS31217 improves the ERG recovery and decreased cell death [45]. INS37217 also reduces subretinal blebs in rabbits [46]. Injection of another P2Y2 agonist, INS542, increases the active transport of fluorescein across the RPE, consistent with this upregulation of ion and fluid transport across the tissue [47]. Together these experiments emphasize the clinical potential of treating retinal edema with P2Y2 agonists.
Regulation of purine levels in subretinal space
While synthesized purinergic agonists may prove useful in treating some ocular disorders, the endogenous activation of P1 and P2 receptors in the subretinal microenvironment will ultimately be determined by availability of agonists. These levels are largely controlled by the release of ATP into the subretinal space, its conversion into other purines including adenosine, and the manipulation of adenosine by enzymes or transporters. Recent work has increased our understanding of both the stimuli that initiate changes in subretinal purine levels and the mechanisms that mediate these changes.
Release of ATP by the RPE
At least some of the ATP capable of stimulating the purinergic receptors on RPE cells is released from the RPE itself. The resulting autocrine stimulation ensures local delivery, and control, of purines to initiate the physiologic changes in the RPE. The release of ATP by RPE cells is triggered by multiple stimuli including osmotic stress, bFGF, UTP, NMDA, glutamate, and ATP [39, 40, 48–51]. The ATP release following activation of NMDA receptors by glutamate may have the most interesting implications for communication across subretinal space, given that glutamate confers the light signal from photoreceptors to the rest of the visual system. Glutamate and the specific receptor agonist NMDA triggers ATP release from ARPE-19 cells, with the release inhibited by NMDA antagonist MK-801, and by DCKA, which inhibits the glycine B binding site on NMDA receptors [51, 52]. Although NMDA raises intracellular Ca2+ levels, this increase is prevented by eliminating ATP with apyrase, indicating autostimulation through released ATP is responsible for this Ca2+ signal. NMDA also triggers a release of ATP when applied to the intact bovine RPE eyecup [51]. The NMDA receptors and the ATP release sites have been functionally identified to the apical membrane of the bovine RPE, suggesting the neurotransmitter interactions could amplify the signal from any glutamate reaching subretinal space.
The ability of both UTP and ATP to stimulate release of ATP from the RPE supports the theory that the system acts to amplify signals. When applied at greater than 1 μM, ATP triggers a secondary release of ATP peaking 10 min after the initial stimuli [40]. UTP also initiates a release in extracellular ATP with a similar delay [48]. The rise in ATP triggered by UTP is inhibited by the Cl- channel blocker NPPB, and UDP is much less effective at triggering release than UTP; both observations suggest the P2Y2 receptor contributes to the increase in ATP more than diphosphokinase, although influence from the enzyme cannot be ruled out [53].
Recent evidence suggests that ischemia may lead to the release of ATP from RPE cells. Chemical ischemia triggers a substantial ATP release from cardiac myocytes [54], while changes in oxygen levels trigger ATP release in central chemoreceptors [55]. We found that exposure to sodium cyanide led to a rapid release of ATP from ARPE-19 cells (Fig. 2). As hypoxic and/or ischemic challenge may lead to changes in the expression of growth factors in RPE cells during certain ocular disorders such as macular degeneration [56], and as purines can induce expression of VEGF in other cells [57], this ATP release may contribute to growth factor signaling by the diseased RPE.
Fig. 2Chemical ischemia triggers ATP release from ARPE-19 cells. ATP release was measured in the bath directly from cells plated in 96-well plates to which the luciferin- luciferase reaction mixture was added [51]. Left Levels of ATP in the bath after addition of 5 mM NaCN to the cells. Measurement began 1 min after addition of NaCN or control solution to wells. Right Levels of ATP measured at the peak, 3 min after addition of NaCN (n = 12). Levels were normalized to concurrent levels in control (n = 14). Symbols and bars represent mean ± SE, *p < 0.001
The particular conduit for ATP release varies with the stimuli. The release in response to hypotonic challenge is largely dependent upon CFTR, as it was prevented by the specific CFTR inhibitor CFTR172 in addition to the more general blocker glybenclamide [50]. While the precise mechanisms by which CFTR contributes to this release are not yet known, a role for CFTR in ATP release into subretinal space is consistent with the reduction of certain ERG components in cftr -/- mice [58] and with the ability of apical ATP to activate conductances associated with these ERG components [43]. The release of ATP is also largely blocked by the vesicular transport inhibitor brefeldin A, suggesting the two processes occurred in series whereby ATP efflux follows the insertion of vesicles containing CFTR into the plasma membrane. Although the Ca2+ chelator BAPTA blocks this ATP release [50], raising Ca2+ alone with ionophore ionomycin does not itself initiate release [48]. This necessary but not sufficient contribution of Ca2+ also supports a role for vesicular insertion.
In contrast to the release following hypotonic challenge, the ATP release in response to NMDA does not involve CFTR [51]. Release is blocked by NPPB, however, suggesting another type of anion channel could serve as a conduit for ATP release. The presence of parallel mechanisms coexisting on the same cell for ATP release triggered by either agonists or by cell swelling has also been reported in astrocytes [59] and may reflect the multiple roles of purinergic signaling within a given tissue. As both stimuli lead to release across the apical membrane into subretinal space, both are expected to influence signaling in the microenvironment.
Interconversion of purines in subretinal space
The interconversion of nucleotides and nucleosides each capable of stimulating distinct receptors makes the purinergic signaling system of particular interest in a confined region such as the subretinal space. The main enzymes responsible for dephosphorylating extracellular ATP on the RPE cells have been analyzed and a basic understanding of their regulation has begun. This section first describes the enzymes that act on ATP and ADP, followed by enzymes which convert AMP into adenosine.
The dephosphorylation of extracellular ATP by RPE cells involves enzymes from multiple families [40], as found in airway epithelial cells [60]. Degradation of ATP by the apical membrane of the fresh bovine eyecup and by ARPE-19 cells is inhibited by ARL67156 or βγmATP. Message for eNPP1, eNPP2, and eNPP3 is present in ARPE-19 cells, consistent with the preference of βγmATP for members of the eNPP family [61]. The cells also express NTPDase2, and NTPDase3, although the intermittent presence of NTPDase1 likely reflects a regulated process [40]. Ecto-alkaline phosphatase has no effect on ATP degradation in RPE cells, in contrast to its considerable contribution in airway epithelium [62]. The putative contribution from diphosphokinases to interconversion of subretinal purines is presently unknown.
Extracellular AMP is rapidly dephosphorylated into adenosine in subretinal space. The production of adenosine from ATP at the apical membrane of the bovine RPE eyecup is inhibited by the ecto-5′-nucleotidase inhibitor αβmADP, confirming a role for this enzyme [63]. The enzyme is localized to rat RPE and ARPE-19 cells immunohistochemically. Degradation of 5′AMP is highest near the subretinal space of rat retina [63], although localization in mouse indicated larger amounts of ecto-5′-nucleotidase at the tips of adjacent Müller cells [64]. Levamisole does not inhibit the dephosphorylation of 5′AMP by the RPE, consistent with the absence of substantial ecto-alkaline phosphatase in subretinal space.
The presence of light may alter the levels of adenosine in subretinal space. Epinephrine is released at the onset of light [65] and stimulation of the RPE with epinephrine can decrease activity of ecto-5′-nucleotidase [63]. While norepinephrine and phenylephrine lead to similar decreases in enzyme activity, prazosin and corynanthine block the effects of norepinephrine, implicating the α1 epinephrine receptor in the inhibition of ecto-5′-nucleotidase [63]. The kinetics of inhibition are consistent with cleavage of the nucleotidase from its GPI anchor. The phagocytosis of rod outer segments is maximal shortly after light onset [16], and this phagocytosis is inhibited by adenosine [29]. The ability of epinephrine released by the illuminated retina to reduce ecto-5′-nucleotidase activity and consequently adenosine levels may relieve this inhibition and enhance the rate of phagocytosis at light onset.
Physiologic effects of subretinal purines on the RPE and photoreceptors
The number of purinergic receptors on both photoreceptor and RPE membranes suggests purines make multiple contributions to the physiology of the outer retina. Our increased understanding of how agonist levels in subretinal space are controlled has begun to indicate how and when this contribution may occur. Future research will involve applying these findings from isolated systems to intact RPE-photoreceptor models, and pursuing the role of defective purinergic regulation in ocular disease. While it is unlikely that ATP released across the apical membrane of the RPE can diffuse to these P2 receptors in the outer plexiform layer given the ecto-ATPase activity in the synaptic clef [25], stimulation of receptors elsewhere on the photoreceptor membrane is possible. It would be interesting to determine whether ATP released from the RPE and converted to adenosine by ecto-nucleotidases can actually modulate the response to light by stimulating the A2A receptors on photoreceptor outer segments. The impact of purinergic signaling on chronic ocular diseases is also of interest, such as the role of ischemia-driven ATP release in VEGF production. While the small size of subretinal space can complicate pharmacologic manipulation within the intact RPE-photoreceptor complex, molecular approaches may provide new insight into how endogenous purines in subretinal space affect the physiology, and pathophysiology, of both RPE and photoreceptors.

rpe
photoreceptors
microenvironment
glutamate
atp release
cftr
ecto-5′-nucleotidase
ntpdase