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Epidemilogical Trends Strongly Suggest Exposures as Etiologic Agents in the Pathogenesis of Sporadic Alzheimer's Disease, Diabetes Mellitus, and Non-Alcoholic Steatohepatitis
  Journal of Alzheimer's Disease 17 (2009) 519-529
DOI 10.3233/JAD-2009-1070
Suzanne M. de la Monte, Alexander Neusner, Jennifer Chu and Margot Lawton Departments of Pathology, Clinical Neuroscience, and Medicine, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI, USA
Supported by AA-02666, AA-02169, AA-11431, AA-12908, and K24-AA-16126 from the National Institutes of Health.
Nitrosamines mediate their mutagenic effects by causing DNA damage, oxidative stress, lipid peroxidation, and pro-inflammatory cytokine activation, which lead to increased cellular degeneration and death. However, the very same patho- physiological processes comprise the "unbuilding" blocks of aging and insulin-resistance diseases including, neurodegeneration, diabetes mellitus (DM), and non-alcoholic steatohepatitis (NASH). Previous studies demonstrated that experimental exposure to streptozotocin, a nitrosamine-related compound, causes NASH, and diabetes mellitus Types 1, 2 and 3 (Alzheimer (AD)-type neurodegeneration). Herein, we review evidence that the upwardly spiraling trends in mortality rates due to DM, AD, and Parkinson's disease typify exposure rather than genetic-based disease models, and parallel the progressive increases in human exposure to nitrates, nitrites, and nitrosamines via processed/preserved foods. We propose that such chronic exposures have critical roles in the pathogenesis of our insulin resistance disease pandemic. Potential solutions include: 1) eliminating the use of nitrites in food; 2) reducing nitrate levels in fertilizer and water used to irrigate crops; and 3) employing safe and effective measures to detoxify food and water prior to human consumption. Future research efforts should focus on refining our ability to detect and monitor human exposures to nitrosamines and assess early evidence of nitrosamine-mediated tissue injury and insulin resistance.
Nitrosamines and N-nitroso compounds are among the most potent and broad acting carcinogens that can also function as transplacental mutagens. Nitrosamine- mediated target organ damage and mutagenesis are heavily influenced by route of administration, dose, chemical nature of the compound, and frequency of exposure. Among the more than 300 N-nitroso or nitrosamine compounds tested, over 90% have been found to be carcinogenic in various organs including liver, gastrointestinal tract (from esophagus to rectum), lung, kidney, bladder, pancreas, prostate, and uterus. All mammals are susceptible. Fried bacon, cured meats, beer, cheese products, fish byproducts, nonfat dry milk, tobacco, gastric juices (dietary), and water are major regular sources of consumer exposure to ni- trosamines. However, nitrosamine exposures also oc- cur through manufacturing, processing, and utilization of rubber and latex products, fertilizers, pesticides, and cosmetics (for review, see [1]).
Nitrosamines (R1N(-R2)-N=O) are formed by a chemical reaction between nitrites and secondary amines or proteins. How does this occur, and what factors contribute to human dietary exposures? First, as a public health measure, sodium nitrite is deliber- ately added to meat and fish to prevent toxin produc- tion by Clostridium botulinum.
Second, sodium nitrite is used to preserve, color, and flavor meats. Heating, acidification, or oxidation of nitrite leads to nitrous acid formation. The resulting nitrosonium cation (N=O+) is a nitrosating agent that reacts with dimethylamine to generate nitrosamines. Dimethylamine is common- ly present in fish meal. Moreover, since ground beef, cured meats, and bacon in particular, contain abundant amines due to their high protein content, and they have significant levels of added nitrates and nitrites [2], ni- trosamines are nearly always detectable in these food products. Finally, nitrosamines are easily generated under strong acid conditions, such as in the stomach, or at high temperatures associated with frying or flame broiling. Reducing sodium nitrite content definitely lowers nitrosamine formation in foods.
Nitrate exposures pose human health problems be- cause about 5% of ingested nitrates get chemically re- duced, forming harmful nitrites by bacterial enzymes present in the oral cavity [3,4]. After entering the high- ly acidic gastric juice environment, nitrites can be con- verted to nitrous acid (HNO 2), and nitrosating agents such as nitrous anhydride (N2O3) and nitric acid [5]. Nitrosating agents [6] and nitrites, either formed en- dogenously or ingested directly with food or water [7] react with amines to generate nitrosamines. Amine precursors of N-nitroso compounds are generated from dietary sources during the natural process of protein digestion. Alternatively, they can be consumed di- rectly when present in overcooked or processed meats and over-ripened fruits and vegetables. High protein content foods yield high levels of amine [8]. Besides amines, choline, a nutrient found in most foods, includ- ing meats, vegetables, peanuts, and eggs, is essential for many biological functions [9], but can react with nitrites to form dimethylamine (DMA), the precursor to nitrosodimethylamine (NDMA) [4].
In essence, diets rich in amines, choline, and nitrates lead to increased nitrosamine production relative to di- ets that are low in nitrate, and include fish and seafood as the main sources of amines [3,10]. Human expo- sure to nitrates stems in part from the abundant use of nitrate-containing fertilizers for agriculture, which re- sults in relatively high levels of nitrates in many crops, particularly root vegetables, such as potatoes and beets. Vegetables, especially potatoes, account for nearly 85% of our daily dietary intake of nitrates [3,6]. Nitrates can also leech from the soil and contaminate water supplies used for crop irrigation, food processing, and drinking. Since amines and choline are essential nutrients, and therefore cannot be eliminated from the diet, a more feasible approach for reducing exposures to nitrates, nitrites, and nitrosamines, would be to discontinue their deliberate addition and permitted contamination of our food and water sources.
The two main forms of nitrosamine that threat- en human health are N-nitrosodimethylamine (DMN; NDMA) and N-nitrosodiethylamine (DEN; NDEA). NDMA and NDEA are highly toxic, semi-volatile, organic chemicals that contaminate food and water sources [11]. NDMA is a waste- or by-product of in- dustrial processes such as organic nitrogen-containing wastewater chloramination, water chlorination, rocket fuel production, and anion exchange resin water treat- ment [12,13]. NDMA can also be found at low levels in tobacco smoke and foods such as cured meat, fish, and beer [2]. NDMA contamination of drinking water and food is problematic because, even at virtually unde- tectable levels, the compound can be harmful, particu- larly after long-term bioaccumulation. Experimentally, short-term, high-level exposure to NDMA is hepato- toxic and causes liver fibrosis [14-16], whereas chronic low-level or moderate exposure to NMDA causes liver tumors [14,17-19]. In humans, nitrosamine exposure from preserved meats and fish has been correlated with increased frequency of gastric cancer [20,21].
NDEA is a volatile water-soluble chemical that is used as an additive for gasoline and lubricants, and in the manufacturing of polymers, plastics, and pesti- cides. Human exposures often occur through inges- tion, inhalation, or skin contact with these substances.
Like NDMA, NDEA contaminates air, food, beverages, drinking water, tobacco smoke, herbicides, and pesti- cides, and is also an industrial pollutant. Appreciable levels of NDEA can be found in inhaled and side-stream tobacco smoke and cured meats. Although NDEA is widespread in the environment, sunlight and UV irra- diation cause it to decompose, thereby reducing expo- sure from inhaled air and open bodies of water [22]. NDEA exposure causes cancer in liver, thyroid, gas- trointestinal tract, and respiratory tract [22]. The main specific carcinogenic nitrosamines associated with ex- posure to tobacco products, including cigarettes, cigars, and chewing tobacco, include N-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [23], the latter of which is produced by reaction of nitrite in saliva with tobacco alkaloids such as nicotine. Since nitrosonornicotine is also present in cigar and cigarette smoke, non-smokers can be exposed via second-hand smoke inhalation. Finally, nicotine replacement prod- ucts can also serve as sources of nitrosamine expo- sure [23]. In essence, discontinued use of tobacco and tobacco-related products may be the single most im- portant means of reducing nitrosamine exposure and related adverse effects on health status.
Nitrosamines exert their toxic and mutagenic effects by functioning as alkylating agents. In brief, metabolic processing converts nitrosamines into active methylat- ing agents that are highly reactive with nucleic acids, al- tering gene expression and causing DNA damage. Ac- tivated nitrosamines, most commonly alkylate N-7 of guanine, lead to destabilization and increased breakage of DNA [17]. In addition, activated nitrosamines gen- erate reactive oxygen species such as superoxide (O 2-) and hydrogen peroxide (H2O2), causing increased ox- idative stress, DNA damage, lipid peroxidation, and protein adduct formation [24]. Oxidative stress and DNA damage lead to activation of pro-inflammatory cytokines, insulin resistance, and aging, all of which are key elements in the pathogenesis of Type 2 diabetes mellitus (T2DM), neurodegeneration, and malignancy. The consequences of nitrosamine-mediated tissue in- jury may vary with the phenotypic properties of the tar- get cells, i.e., proliferating cells may be more prone to undergo malignant transformation, whereas terminally differentiated cells may instead undergo senescence or degeneration.
Investigations about the role of nitrosamines in the pathogenesis of human disease have primarily focused on carcinogenesis, yet the basic cellular and molecu- lar alterations produced by nitrosamine exposure are fundamentally similar to those that occur with aging, Alzheimer's disease (AD), non-alcoholic steatohepati- tis (NASH), and T2DM. All of these non-neoplastic dis- eases are associated with increased insulin resistance, DNA damage, lipid peroxidation, oxidative stress, and pro-inflammatory cytokine activation [25-31]. The prevalence rates of AD, NASH, obesity, and T2DM have all increased exponentially over the past several decades, and thus far, show no hints of plateau [32-37]. Of particular note is that the rather short time inter- val associated with dramatic shifts in disease incidence and prevalence rates is more consistent with exposure- related, rather than genetic etiologies (discussed later).
An important clue regarding the probable connec- tion between nitrosamine exposure and AD, NASH, and diabetes mellitus was provided by experimental da- ta demonstrating that treatment with Streptozotocin [2- deoxy-2-(3-methyl-3-nitrosoureido-D-glucopyranose (C8H15N3O7)] (STZ), a glucosamine-nitrosourea compound and derivative of N-methyl-N-nitrosourea (MNU), causes AD-type neurodegeneration with cog- nitive impairment, T1DM, T2DM [38-44], or hepatic steatosis with chronic inflammation and scarring, i.e., NASH [45]. Of further interest is the finding that sim- ilar disease models can be produced by delivery of ei- ther a single large dose (40-60 mg/kg) [43], or repeated smaller doses [42] of STZ. STZ is taken up by cells via glucose transporters [46], and once metabolized, liberates N-nitrosoureido. STZ inhibits DNA synthesis and kills cells by lowering ATP and nicotine adenine dinucleotide (NAD+) content [43].
Structurally, STZ is quite similar to nitrosamines, and like other N-nitroso compounds, including NDEA and NDMA [47], STZ's MNU causes cellular injury and disease by functioning as: 1) an alkylating agent and potent mutagen resulting in cancer development in various organs [48]; 2) an inducer of DNA adducts, most significantly N7-methylguanine, which lead to in- creased apoptosis [49]; 3) a mediator of unscheduled DNA synthesis that triggers cell death [48]; 4) an in- ducer of single-strand DNA breaks; 5) a stimulus for nitric oxide (NO) formation following breakdown of its nitrosamine group [43]; and 6) an enhancer of the xan- thine oxidase system leading to increased production of superoxide anion, H2O2, and OH - radicals [50]. In essence, STZ-induced cellular injury is mediated by the generation of reactive oxygen species with atten- dant increased levels of superoxide, nitric oxide, and lipid peroxidation, all of which cause DNA damage. Radical ion accumulation leads to inhibition of oxida- tive metabolism, mitochondrial dysfunction [43], de- creased ATP production [51], activation of poly-ADP ribosylation, and finally cell death.
STZ treatment causes T1DM [44,52], T2DM [53- 55], steatohepatitis [45], and neurodegeneration [38- 41]. The spectrum of disease produced is dictated by the dose and route of administration of STZ. In- traperitoneal delivery of STZ causes T1DM, T2DM, or NASH, with high doses usually resulting in T1DM, and lower repeated doses causing T2DM or steato- hepatitis. Intracerebral (ic) STZ administration causes neurodegeneration in the absence of T1DM, T2DM, or NASH, indicating that the brain can be a selective target of MNU/nitrosamine-mediated injury or neu- rotoxicity. Importantly, these disease entities share a common theme, but vary with respect to nature and degree of target-organ injury, insulin deficiency and/or insulin resistance, increased oxidative stress and DNA damage, increased levels of nitric oxide and free radicals, pro-inflammatory cytokine activation, and mitochondrial dysfunction with deficiencies in ATP production. Specific effects of ic-STZ-induced neu- rodegeneration include: increased amyloid-ß protein precursor-amyloid-ß (AßPP-Aß) deposition, AßPP gene expression, and tau phosphorylation, accompa- nied by cognitive impairment [38,41,56], deficits in acetylcholine homeostasis [38,41] and neurotrophin gene expression [57], as typically occur in AD [58]. Since the cognitive deficits and neurodegeneration caused by ic-STZ can be abrogated by concurrent delivery of insulin [59], or early intervention with peroxisome-proliferator activated receptor (PPAR) ag- onists, which function as both insulin-sensitizer and anti-inflammatory/antioxidant agents [38], this AD model mechanistically overlaps with both T1DM and T2DM, prompting us to coin the term, "Type 3 Dia- betes" [38,41]
In essence, at the core of AD pathology, DM, and NASH is insulin resistance with associated deficits in glucose utilization and energy metabolism, and in- creased levels of chronic inflammation and oxidative stress [60-65]. Although for years, disturbances in metabolic function have been recognized features of both DM and AD [60], the mediators of these abnor- malities were not understood. However, the more re- cent and growing interest in the roles of insulin resis- tance and insulin deficiency syndromes, including AD, has opened doors to more diversified investigational approaches. Correspondingly, epidemiological trend analysis demonstrated significant associations between AD and T2DM, revealing the nearly two-fold increased risk of developing AD in subjects with T2DM [25,65- 71]. In addition, other studies showed associations be- tween obesity or peripheral insulin resistance and cog- nitive impairment [72,73]. Since NASH and AD are associated with T2DM, T2DM and NASH are associat- ed with obesity and metabolic syndrome, while AD and T1DM are associated with insulin deficiency, there ap- pears to be a 3-way Venn-diagram type overlap among AD, T2DM, and NASH. But, is there a common cause?
We were prompted to consider the potential role of nitrosamines as environmental toxins mediating insulin-resistance diseases, including NASH, T2DM, and AD because STZ has a methylnitrosourea group and its effects on DNA and cellular functions are similar to those of nitrosamine compounds, including NDEA and NDMA (Fig. 1). However, since in low, sub- mutagenic doses, STZ causes insulin resistance, we considered the concept that low-dose chronic expo- sure to NDEA, NDMA, and other nitrosamines may cause insulin-resistance mediated diseases rather than malignancy. To assess the validity of an exposure hy- pothesis in relation to the pathogenesis of T2DM and AD, it was first necessary to demonstrate epidemio- logical trends that mirrored effects of exposure-related diseases such as infectious diseases, rather than genet- ic diseases which remain relatively stable over a peri- od of decades. Therefore, we graphed and analyzed time-dependent, age-stratified mortality rates from all causes, AD, Parkinson's disease (PD), diabetes melli- tus, cerebrovascular disease, chronic liver disease, lym- phomas, leukemias, and HIV-AIDS from 1968 to 2005. HIV-AIDS served as a positive control for disease that is caused by exposure to an infectious agent. The da- ta source was the US Department of Health and Hu- man Services, Centers for Disease Control and Pre- vention National Center for Health Statistics database ( To first determine the degree to which death rates from these diseases corre- lated with increasing age, we computed areas under the curves corresponding to death rates over time versus age group, i.e., 45-54, 55-64, 65-74, 75-84, and 85+ years (Fig. 2). The graphs demonstrate that the highest rates of death from nearly all of these disease occurred in the older age groups, indicating that they are aging- associated. Exceptions included chronic liver disease and HIV-AIDS, which had higher death rates in the earlier age groups.
To determine the degree to which the mortality rates had shifted upward or downward over time, we graphed age-stratified death rates for each disease. This ap- proach enables direct comparison of death rates from any given disease in, for example, 65-74 year olds in 1970 versus the same age group in 2005. Moreover, by superimposing or aligning graphs for all age groups within a disease category, it was possible to determine the rank order of death rates with respect to age group (Fig. 3). However, to clearly visualize specific disease trends, the death rates corresponding to individual age brackets were re-plotted to scale on separate graphs (Fig. 4). Among individuals 45-54, 55-64, 65-74, 75- 84, and 85+ years old, the death rates from all causes progressively declined over time (Fig. 3A). Similarly, death rates from cerebrovascular disease (Figs 3E, 4A- 4E) and chronic liver disease (Fig. 3F) also declined within each age group over time. Nonetheless, for each of these diseases, the death rates were highest among the 85+ group followed by the 75-84 year olds, while the other three groups were lower but tightly clustered. In contrast, the trends for lymphomas and leukemias remained relatively stable (Fig. 3G, 3H), except for the time-dependent upward drift in death rate from lym- phoma among 85+ year olds (Fig. 3G).
Death rates from HIV-AIDS exhibited classical exposure-associated increases from 1985 to 1995, fol- lowed by precipitous declines due to increased avail- ability of effective anti-retroviral drugs (Figs 3F, 4F- 4J). Trends mapping death rates from DM were U- shaped for all age groups in that the rates sharply de- clined after 1968, reaching a nadir in -1980, but subsequently increased to levels that were similar to, or higher than in 1968, with modest hints of plateau over the last 3-4 years (Figs 3D, 4K-4O). Death rates from AD (Figs 3B, 4P-4T) and PD (Figs 3C, 4U-4Y) exhib- ited opposite trends relative to cerebrovascular disease and all causes of death due to increased rather than de- creased rates within each age group over time. Of note is that between 1968 and 2005, the death rates from AD increased by 4-fold in the 55-64, 20-fold in the 64-74, 150-fold in the 75-84, and 800-fold in the 85+ year old groups. With respect to PD, the death rates increased about 2-fold in the 65-74, 3-fold in the 75-84, and near- ly 6-fold in the 85+ age groups from 1980-2005 (PD death rates were not tracked prior to 1980). Therefore, besides bucking the trends with respect to other major aging-associated diseases, the relatively short intervals associated with dramatic increases in death rates from DM, AD, and PD are more consistent with exposure- related rather than genetic etiologies. Moreover, the strikingly higher and climbing mortality rates in older age brackets suggest that aging and/or longer durations of exposure have greater impacts on progression and severity of these diseases. The U-shaped curve for DM is particularly disconcerting because presumably the recognition and treatments are better today than they were in 1980, yet mortality is higher. Perhaps factors contributing to the pathogenesis of DM prior to 1970 differ from those in modern times,which seem to render DM a more "malignant" and uncontrollable disease. Since human exposures to nitrates, nitrites, and ni- trosamines are likely to occur due to fertilizer use, con- sumption of processed and preserved foods, and agri- cultural products that heavily rely in fertilizer use (e.g., grains), we examined US population growth (Fig. 5A), annual use/consumption of nitrate-containing fertiliz- ers (Fig. 5B), annual sales for a popular fast food fran- chise (Fig. 5C), sales for a major meat processing com- pany (Fig. 5D), consumption of grain (Fig. 5E), and consumption of watermelon and cantaloupe (Fig. 5F). Watermelon and cantaloupe served as controls since they are not typically associated with nitrate or ni- trite exposure, DM, or AD. Business sales data were obtained from the companies' websites or stock ex- change databases, and agricultural sales/consumption data were obtained from USDA websites. The results demonstrated that the US population nearly doubled between 1955 and 2005. Although nitrogen- containing fertilizer consumption increased by 230% over the same interval, its usage doubled between 1960 and 1980, just preceding the insulin-resistance epidemics. Sales from a popular fast food franchise and a major meat processing company increased more than 8-fold from 1970 to 2005, and grain consumption increased 5-fold. In contrast, watermelon consumption remained relatively flat, while cantaloupe consumption nearly doubled, paralleling population growth, although over- all per capita consumption remains relatively low com- pared with processed foods.
Epidemiological data revealed staggering growths in prevalence and/or mortality rates for T2DM, obesity, NASH, and AD, and significant overlap between AD or NASH and T2DM. Basic, translational, and clinical research studies have demonstrated that AD neurode- generation, like T2DM and NASH, is associated with insulin resistance as well as increased oxidative stress, DNA damage, NO-mediated injury, and ATP deficits caused by mitochondrial dysfunction and impaired in- sulin signaling, but with prominent or selective involvement of the brain. Experimental animal models pro- vided evidence that STZ administration causes Type 1, Type 2, or Type 3 diabetes, depending on dose and route of administration. In addition, STZ treatment causes hepatic steatosis associated with insulin resis- tance, pro-inflammatory cytokine activation, increased oxidative stress, DNA damage, lipid peroxidation, and cell death culminating in fibrogenesis, i.e., a model of NASH [48,74].
Returning to the epidemiological data, the time course of increasing prevalence rates of T2DM, NASH, and AD cannot be explained on the basis of gene mutations, and instead, mirror the classical trends of exposure-related diseases. Since STZ is structurally re- lated to nitrosamines and produces similar biochemical and molecular lesions in cells and tissues, it is conceiv- able that chronic exposure to relatively low levels of ni- trites and nitrosamines through processed foods, water, and fertilizers, is responsible for our current epidemics and probably also the pandemics of T2DM, NASH, and AD. The link to obesity is through excessive consump- tion of nutritionally irresponsible processed foods. If this hypothesis is correct, potential solutions would in- clude: 1) eliminating the use of nitrites and nitrates in food processing, food preservation, and agriculture; 2) taking steps to prevent formation of nitrosamines; and 3) employing safe and effective measures to detoxify food and water prior to human consumption. In addi- tion, further development of highly sensitive measures to detect the footprints, e.g., measures of amine nitrosa- tion, and assess levels of nitrosamine exposure in body fluids such as urine or cerebrospinal fluid, and in tissues such as liver, brain, adipose tissue, and muscle, could help identify individuals at risk for developing T2DM, NASH, and/or AD.
Nitrosamines are potent mutagens that cause DNA damage, oxidative stress, cell death, and cancer. How- ever, the structural relatedness of nitrosamines to STZ demands attention because STZ is a proven mutagen, but in low doses causes Types 1 and 2 diabetes mel- litus, hepatic steatohepatitis, or AD-type neurodegen- eration. We hypothesize that widespread exposures to nitrites and nitrosamines in our environment have criti- cal roles in the pathogenesis of these insulin-resistance and insulin-deficiency related diseases. Epidemiolog- ical trends support exposure rather than genetic caus- es of these diseases, and exposures to nitrites and nitrosamines through food, water, agriculture have in- creased just prior to and within the same interval due to proliferation of food processing, increased require- ment for food preservation to enable "safe" storage and long distance shipping, and the use of fertilizers to enhance crop growth and meet growing demand for produce. Tobacco use contributes to the problem, but public health measures have already been taken to re- duce public exposure. Sincere efforts should be made to significantly curtail or eliminate human exposure to nitrates and nitrites, and re fine extant biotechnology to monitor exposure, metabolite formation, and asso- ciated cellular and tissue injury linked to nitrosamine- mediated insulin resistance-related diseases, including T2DM, NASH, and AD.
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