Chapter 35 Environmental Medicine
Environmental Toxic Load
The twentieth century, with its promise of “Better Living Through Chemistry,” brought us a host of chemical toxicant-related illnesses (referred to here as “environmental illnesses”). We are experiencing the new medical diagnoses of sick (closed) building syndrome1,2 and multiple chemical sensitivity,3–5 both of which are known to be related to overexposure to environmental contaminants. The rates of asthma, allergy, autism, attention deficit hyperactivity disorder, and childhood brain tumors, obesity, and diabetes are all skyrocketing. All of these have been linked to the growing environmental burden that is carried by all persons. The primary physiologic action of the major pesticide classes disrupts neurologic function.6 The primary physiologic action of solvents is neurotoxicity7 as well. In addition to being neurotoxic, these compounds are profoundly immunotoxic8–10and are often toxic to the endocrine system as well.11–13 The adverse health effects are not limited to those systems as these compounds can also cause a variety of dermatologic, gastrointestinal, genitourinary, respiratory, musculoskeletal, and cardiologic13–15 problems. A great many of the adverse effects of these environmental toxins appear to be secondary to their mitochondrial damaging capacity.
The Toxic Burden
In an effort to identify what the full burden of environmental chemicals in the U.S. population is, the Centers for Disease Control (CDC) has been performing an ongoing assessment of the levels of environmental chemicals in the U.S. population. This ongoing study utilizes laboratory samples from persons involved in the National Health and Nutrition Examination Survey from the years 1999 to 2000, 2001 to 2002, and 2003 to 2004 (each representing about 2400 persons). In the First National Report on Human Exposure to Environmental Chemicals, 27 different compounds were measured.16 They tested for the presence of 13 different heavy metals, cotinine, six different organophosphorus pesticide (OP) metabolites, and seven phthalates and their metabolites. Nine of the heavy metals (including lead and mercury), three of the organophosphorus metabolites, and three of the phthalate metabolites appeared fairly ubiquitously. The second report was published 2 years later and expanded its scope of measurement by an additional 89 compounds for a total of 116 chemicals for the years 1999 and 2000.17 The second report included measurements of dioxins, furans, polycyclic aromatic hydrocarbons (PAH), herbicides, carbamate pesticides, certain organophosphate pesticides (including chlorpyrifos), phytoestrogens, and organochlorine pesticides. Of the 14 PAHs measured (all from combustion) five were found ubiquitously. In the third report (2005), pyrethroid insecticides were added to the list.18
In the fourth report, 75 new compounds were measured, bringing the grand total of measured xenobiotics to 212.19 This report included blood and urinary levels of eight different forms of arsenic along with several aromatic solvents (a total of 30 different compounds) for the first time. This was also the first of the reports that gave values for mercury in adults rather than juveniles and showed that the majority of mercury in the blood is organic rather than elemental. Acrylamides, trihalomethanes (from water disinfection), bisphenol A, phthalates, triclosan, polybrominated diphenyl ethers (flame retardants), benzophenone from sunblock, perfluorocarbons from nonstick coatings, and a host of other xenobiotics were found in the majority of persons tested in the fourth report. A review of the pertinent findings of the fourth report is available online.20
The Environmental Working Group (www.ewg.org) has funded and published two studies that specifically tested adults and newborns in the United States for xenobiotic burden. They originally tested 9 adults for the presence of 210 environmental toxins.21 These nine adults had an average of 91 of the 210 toxic compounds. Because these compounds can also be passed from mother to child, the EWG did a study to examine how many toxins were passed to babies. The EWG newborn study tested for 423 toxic chemicals in the cord blood of 10 infants born in U.S. hospitals in the year 2004.22 A total of 287 toxic compounds were found in the samples of the cord blood, with an average of 200 per child. One hundred one of these toxic compounds were found in all of the samples, with a range of total chemicals from a low of 154 compounds per child to a high of 231.
These studies, and hundreds more that have been published in the medical and scientific journals, have shown that we are all burdened with numerous toxic compounds. Examples include the fat-soluble polychlorinated biphenyls (PCBs), chlorinated pesticides (dichlorodiphenyltrichloroethane [DDT], etc.), dioxins and furans, plastics, heavy metals, OPs, aromatic hydrocarbons from tailpipes and cigarettes, flame retardants, napthalenes, and perfluorocarbons. It is no longer a question of if we are all burdened with toxic compounds, but rather if this burden is a causative factor in our health complaints and an obstacle to cure.
Sources of Environmental Toxins
In the previous section, the EWG newborn study22 highlighted the fact that mothers unwittingly pass fat-soluble toxins to their child during gestation. After birth, the toxic exposures primarily come from the air in our homes and the food that we consume. The U.S. Environmental Protection Agency did a number of total exposure assessment methodology studies in the 1980s, which showed that indoor air contains higher levels of environmental chemicals than outdoor air (even in towns with multiple chemical plants).23 The majority of compounds found in the indoor air were solvents that came from smoking, dry cleaned clothes, home furnishings, and home cleaning agents. Pesticide use in the home and garden are also contributing factors to the burden of pollutants in indoor air and dust.24,25
Food is the other route through which the majority of our xenobiotic load is delivered. The U.S. Food and Drug Administration (FDA) has an ongoing total diet survey that measures a set list of foods for a variety of environmental contaminants, including heavy metals and plasticizer compounds.26,27 The U.S. Department of Agriculture (USDA) has also been doing an ongoing measurement of pesticide residues on the most commonly consumed fruits and vegetables.28 The data published in this USDA report form the basis for the “dirty dozen” list of the most toxic fruits and vegetables, which are published on the web by the EWG.29 This listing gives the consumer information on how to avoid their greatest exposures to OPs.
Plasticizers contaminate food from simple contact with plastic food wrap. The movement of plastics from the wrapping to the food increases with the length of time they are in contact with each other, the fat content of the food, and whether or not heat is involved. Microwaving foods in plastic wrap increases migration, depending upon how closely the food is in contact with the wrapping.30 Levels of plasticizers are high in store-wrapped meat, poultry, fish, and cheese, with cheese having the highest level of plasticizers. Contrary to a current internet myth, freezing food in plastic containers does not increase plastic levels in the food. Plastic levels in unheated plastic-wrapped foods only increased in those foods after they were heated.31 Our exposure to some plasticizers, however, comes primarily from fragrances and other personal care products.32
Butter has been used as a sampling agent to assess the regional and global distribution of PCBs and other persistent organic pollutants around the globe. When butter was sampled for these compounds from around the world, the highest levels of PCBs were found in butter from Europe and North America.33 For individuals living in North America, the greatest source of PCB exposure came from butter and fish.34 However, by far, the single greatest source of PCBs and other “halogenated” persistent toxins in food is farmed Atlantic salmon from grocery stores and restaurants. A study done by the Food Safety Authority of Ireland found that farmed salmon had an average of four times the amount of PCBs and dioxins as wild salmon.34a Another study revealed that these farmed salmon had PCB levels over five times higher than those found in a sampling of wild salmon.35
These studies were done on relatively small numbers of fish, but had consistent findings. Their findings were confirmed in a subsequent study done on over 700 salmon, totaling approximately two metric tons of farmed and wild salmon from around the globe.36 Thirteen persistent chlorinated chemical pollutants, including the flame retardant polybrominated diphenyl ether, were found in significantly higher levels in farmed salmon than in wild salmon.
Health Effects of Environmental Toxins
Numerous studies have been published on the adverse health effects of individual xenobiotics. Although the areas of impact cover all the systems in the body, this chapter focuses on the documented immunotoxic, neurotoxic, and endocrinotoxic effects of these chemicals.
Of the three major systems affected by xenobiotic burden (immune, neurologic, and endocrine), the signs and symptoms of immunotoxicity are very often the first to occur in a patient’s history. This often begins with the appearance of allergies (including adverse food reactions), after which chemical sensitivity begins. Chronic infections (due to cell-mediated immune dysfunction) and autoimmunity can also be present.8,9,37 Rarely will a single individual manifest all of these, but clinicians are well advised to be vigilant in looking for such a constellation.
Cell-Mediated Immune Dysfunction
Unfortunately, many of the xenobiotics that have been identified inside all of us are very potent suppressors of cell-mediated immune responses. Dichlorodiphenyldichloroethylene (DDE) (the main metabolite of DDT) causes apoptosis of peripheral blood mononuclear cells, resulting in fewer macrophages roaming the body to phagocytize invading pathogens and trigger an immune response.38,39 Persons with the highest levels of DDE in their blood also showed reduced mitogen-induced lymphocyte response to concanavalin A.40 Individuals with this commonly found lipophilic toxin could easily have compromised cellular immune responses. Mercury, another commonly found xenobiotic, also increases apoptosis of both monocytes and lymphocytes and reduces phagocytic ability of the monocytes. It has been demonstrated that workers occupationally exposed to mercury vapor exhibited diminished capacity to produce both tumor-necrosis factor-α and interleukin-1.41
The chemicals produced by combustion, PAHs, have been shown to have similar depressing effects on the immune system, including decreased T-cell dependent antibody response, decreased splenic activity, diminished T-cell effector functions, suppression of T-cell cytotoxic induction, and lower natural killer cell activity.42 The OPs, which are not as biologically persistent as chlorinated pesticides, are also toxic to the immune system. They have been found to cause decreased percentages of CD4 and CD5 cells, increased number and percentages of CD26 cells, increased incidence of atopy and antibiotic sensitivity, and high rates of autoimmunity. This elevation in autoimmunity is reflected by high levels of antibodies to smooth muscle, parietal cells, brush border, thyroid, myelin, and elevated antinuclear antibody (ANA).43
Allergy and Asthma
Increased rates of asthma and allergy have been clearly linked to outdoor air pollution.44 A day or two after ambient ozone levels spike, persons with asthma have exacerbations of their breathing problems.45 Hikers who encounter elevated levels of ozone also experience reduced lung function. With each additional 50 ppb of ambient ozone levels, hikers experience a 2.6% decline in forced expiratory volume and a 2.2% decline in forced vital capacity.46 When persons with asthma and allergies encounter diesel exhaust particles, their allergic problems increase.47 Even those individuals without a history of allergies can become allergic when exposed to diesel emissions at the same time as an allergen. Herbicide and pesticide exposures,48 as well as exposure to phthalates (plasticizers) in house dust have also been associated with increased risk for asthma.49
The development of autoimmunity has been linked with chemical exposure as well. The notion of chemically-induced autoimmune states is, of course, not new because many chemicals are known to induce the onset of systemic lupus erythematosus. Some chemicals, like formaldehyde and other volatile organic compounds, are thought to induce tissue-specific autoimmune reactions by acting as haptens. These low molecular weight molecules bind to various tissues in the body, making a new antigenic combination. The immune system then makes an antibody to this new combination that can attack the parent tissue with or without the chemicals being present. Chemically exposed individuals will often present with elevated antibodies to certain body tissues, including antimyelin, antiparietal, anti-brush border, and antismooth muscle.50 A study of 298 patients with exposure to industrial chemicals revealed several immunologic abnormalities, including autoantibodies against smooth muscle (odds ratio [OR] 3.99), parietal cells (OR 9.7), and brush border cells in the small intestine (OR 14.45), the thyroid, and myelin sheathing.51
Autoimmune hypothyroidism is becoming a much more commonly seen problem in physicians’ offices and can be secondary to exposures beyond just OPs.52 The presence of elevated levels of antinuclear antibodies has also been associated with pesticide exposures other than organophosphates.53
A number of investigators have reported that mercurials are also capable of immune activation, leading to autoimmunity while simultaneously reducing the cellular immune response leading to increased infection,54–57 which is the classic appearance of immunotoxicity.58 In genetically susceptible mice, the presence of mercury dramatically increased their autoimmune response with increased antifibrillarin antibodies.59
The neurologic system is also a frequent target for xenobiotic compounds. Some patients will present primarily with neurotoxicity symptoms, whereas others may exhibit immunotoxicity signs and symptoms first. The most common neurotoxicity symptoms include reduced cognitive functioning (often referred to by the patient as “brain fog” or “crooked brain”), headache, memory problems, and mood disorders. Tremors, balance problems, and anxiety can also be present (Box 35-1). Although there are indications that xenobiotics may play a role in certain neurologic illnesses, Parkinsonism is the illness that shows the most association.
BOX 35-1 Neurotoxicity Presentations
Chronic Low-Level Chlorinated Pesticides
The nervous system is a unique target for toxic agents in several ways:
1. The adult neuron does not divide and, therefore, replacement of lost neurons is not possible. Nerve cells killed by toxins cannot regenerate.
2. The blood–brain barrier does not block nonpolar substances or items that are actively transported.
3. Because the normal function of the nervous system requires the action of a complex integrated network, damage to even a small portion of the nervous system sometimes can result in marked effects on function.
4. Neurons are dependent on glucose and oxygen, and some cell bodies exist at borderline levels of oxygen. If high energy demands are placed on the system and delivery of oxygen is reduced, then cell death may occur.
5. Because of high lipid content (myelin), there is an accumulation and storage of lipophylic xenobiotics.
6. Neurons have high surface areas and, therefore, increased exposure to toxins.
Unfortunately a great many of the common xenobiotic toxics in our bodies are potent neurotoxins. All of the major classes of pesticides kill pests by virtue of their neurotoxic actions. Chlorinated pesticides and pyrethroids disrupt the ion flow along the axon, whereas organophosphates (which came out of nerve gas research in Germany between the first and second world wars60) and carbamates are potent acetylcholinesterase inhibitors (resulting in excessive acetylcholine levels in the synaptic clefts). Solvents, some of which were originally used as anesthetics, dampen the propagation and transmission of electrical impulses along the nerve axons. All of these agents produce various forms of toxic encephalopathy (acute or chronic, selective, or diffuse toxic encephalopathies). Many environmentally ill patients present to their physicians with chief complaints that fit this diagnostic category.
The average clinician will rarely see an individual with acute organophosphate poisoning. Instead, they will see persons presenting with depression, headache, fatigue, cognitive issues, and other problems that could tend to be attributed to other issues of aging. The following OP worker studies highlight these problems.
Greenhouse workers who were exposed to OPs exhibited higher incidences of depression, headache, tremors, and paresthesias.61 Polish female greenhouse workers exposed to OPs exhibited longer reaction time and reduced motor steadiness than unexposed workers. They also reported increased tension, depression, and fatigue more than controls reported.62 Dutch farmers and gardeners who used OPs frequently had much higher risk of developing mild cognitive dysfunction than others.63 Farmers repeatedly exposed to OPs from sheep dip showed much greater vulnerability to psychiatric disorders than controls (quarry workers). They also performed worse than controls on cognitive testing that assessed attention span and how fast they processed information.64 None of the persons in any of these studies fit the definition of acute OP toxicity and none had dramatic reductions of acetylcholinesterase.
Neurologic problems from OP exposure can also persist. A study of persons previously poisoned by OPs revealed many abnormalities, including memory, abstraction, intellectual functioning, mood, and motor reflexes. They also had greater distress and complaints of disability.65 A different study of OP workers also revealed diminished memory, learning, and vigilance, but also found diminished plantar and ankle reflexes.66 The authors noted that the persons with these problems had similar acetylcholine levels as those who did not exhibit these differences. Abnormal deep tendon reflexes were found, along with diminished coordination and muscle strength in Ecuadorian persons exposed to OPs.67
Because the use of chlorinated pesticides has been banned in the United States since the 1980s, the symptomatic picture of acute poisoning with them will not be presented. Instead, we find a picture of generalized neurologic dysfunction very similar to what was just presented for OPs.
Early controlled trials of airborne exposure to low levels of DDT revealed that exposed subjects would experience neurologic symptoms, including dimming of vision, a drawing sensation at the base of the nose or behind the eyes, a sense of fullness deep inside the skull, headache, slowness of thought, inability to concentrate, and short-term memory loss.68 Various muscle symptoms also occurred, including weakness, fatigue, dysphagia, and ataxia. Demonstrable reductions in electromyographic readings were found after nasal exposure to DDT that might account for such symptoms.69
A study that compared retired malaria control workers with a reference group of retired persons who did not handle DDT revealed that the exposed group had significantly poorer performance on cognitive, sensory, and motor testing. They did particularly poorly on the cognitive testing (verbal attention, visuomotor speed, and sequencing) and reported significantly more psychiatric symptoms than controls reported.70
Neurologic effects of chlordane exposure were studied in a group of persons who lived in an apartment complex where chlordane was used. Seven years after the application occurred, residents and former residents of the complex were assessed. Significant changes were found in the exposed persons, including reduced reaction time, balance dysfunction (shown by increased sway speed), reduction in cognitive function, perceptual motor speed, and immediate and delayed verbal recall. They also had worse scores for mood, including increased tension, depression, anger, and fatigue.71
Chronic toxic encephalopathy (CTE) from solvent exposure will often gradually improve in 50% of cases with elimination of solvent exposure (Box 35-2). However, 50% will not improve from mere avoidance in the time frame of 6 to 42 months after initial assessment. It is interesting to note that in one study persons on antidepressants were nearly four times more likely to have persistent CTE than those not on the medications.72 Although the study failed to list which antidepressants were used, many of the antidepressant medications were known to be inhibitors of the CYP system in the liver. Workers with a genetic polymorphism affecting glutathione production had a dramatically increased risk for the development of CTE.73 It is therefore possible that the reason for such poor recovery from CTE after leaving the main source of solvent exposure is the rate at which the liver can clear these compounds from the circulation. In persons with CTE, brain atrophy was noted in over 50% of them using computed tomographic scan.74
BOX 35-2 Chronic Toxic Encephalopathy (CTE): Designations of Solvent-Induced Neurotoxicity
1. Affective syndrome: neuropsychiatric symptoms, no signs of impairment, reversible.
2. Mild chronic toxic encephalopathy: neuropsychiatric symptoms, proven impairments, uncertain reversibility.
3. Severe chronic toxic encephalopathy: more severe neuropsychiatric symptoms, more pronounced impairments, usually irreversible.
Data from World Health Organization meeting on Organic Solvents, Copenhagen, Denmark, 1985.
Toluene, an aromatic solvent that is commonly present in glues and paints, leads to a very classic presentation of CTE. In one study with toluene-exposed printers, the following symptoms were found: fatigue 80%, impaired memory 60%, impaired concentration 40%, irritability 37%, headaches 30%, mood lability 27%, and depression 20%.
Shipyard painters were found to have significantly higher scores for neurotic behaviors than controls reported.75 They were also found to have significantly greater problems with short-term memory, concentration, fatigue, dizziness, and insomnia. They also noted more trouble with a feeling of pressure in the chest and perspiration without work. One of the questions most frequently answered affirmatively by the group (in significantly higher levels than controls) was, “Do you often have to go back to check things that you have done, such as turned off the stove, locked the door, etc?”76
Female workers exposed to toluene showed significantly more problems with manual dexterity, visual scanning, and verbal memory.77 The authors of this study noted that the workers who exhibited these changes on neurobehavioral testing showed absolutely no clinical signs of toxicity!
Heavy Metal Neurotoxicity
Lead is a well-recognized neurotoxin leading to multiple problems in children, including reduced IQ scores,78 and attention79 and behavioral problems. Parents of children with lead burdens report that their children have more somatic complaints and delinquent, aggressive, internalizing, and externalizing behavior. Their teachers report that the children have more problems with anxious/depressed behavior, social problems, attention problems, and delinquent, aggressive, internalizing, and externalizing behavior.80 In Yugoslavian children, specific intelligence testing revealed that lead was most damaging to perceptual–motor aspects of intelligence rather than language-related aspects.81 Lead exposure in children may also be a factor in pervasive developmental disorders, including autism.82 A number of studies have also clearly linked lead burden in children and attention deficit hyperactivity disorder.83–85
When persons who were lead exposed in childhood were studied 20 years later, neurologic deficits were still found. In this group, significant adverse central and peripheral neurologic effects were present. Peripheral nerve function was altered, as were measures of coordination, reaction time, dexterity, learning, and mood.86 As persons with lead exposure grew older, their memory capacity continued to decline, at rates faster than aging could account for.87
Lead workers have consistently shown neuropsychologic problems at levels significantly higher than those of controls. These problems include visuospatial, visuomotor, comprehension, and symptoms of depression, insomnia, fatigue,88,89 and postural sway.90,91 When bone lead burden was measured in older individuals, none of whom had industrial exposures to lead, a clear association was correlated with mental function. As the total bone lead (measured by fluoroscopy of the tibia) increased, cognitive function decreased in a group of older individuals from Baltimore.92
Recently, lead burden has been associated with increased risk for the development of Parkinsonism.93
Mercury in both organic and inorganic forms is neurotoxic. Methylmercury accumulates in the brain and is associated with mitochondria, endoplasmic reticulum, Golgi complex, nuclear envelopes, and lysosomes. In nerve fibers, methylmercury is localized primarily in myelin sheaths, where it leads to demyelination.94 Mercury is also known to inhibit the uptake of dopamine,95 serotonin,96 and norepinephrine97 at synaptic sites. Mercury apparently has a higher binding affinity for serotonin-binding sites.
The widespread pollution of Minamata Bay, Japan, by methylmercury in the 1950s provided researchers with a clear picture of methylmercury-induced neurotoxicity. Known as Minamata Disease, the neurotoxic signs included ataxia, speech impairment, constriction of visual fields, hypoesthesia, dysarthria, hearing impairment, and sensory disturbances. These neurologic problems persisted and were found in other areas of Japan as the mercury contamination spread through fish consumption.98 Follow-up studies in the Minamata area almost 40 years after the spill and almost 30 years since a fishing ban was enacted for the area showed the persistence of mercury neurotoxicity. Residents in fishing villages in the area in 1995 reported significantly higher prevalences than “town resident controls” in males for the following complaints: stiffness, dysesthesia, hand tremor, dizziness, loss of pain sensation, cramping, atrophy of the upper arm musculature, arthralgia, insomnia, and lumbago. Female residents of the fishing villages had significantly higher incidents of complaints of leg tremor, tinnitus, loss of touch sensation, leg muscular atrophy, and muscular weakness.99
In the Amazon, children exposed to methylmercury from local gold mining have also been studied for the neurotoxic effect of methylmercury. In the villages studied, more than 80% of the children had hair mercury levels above 10 mcg/g (a level above which adverse effects on brain development are likely to occur). Neuropsychological tests of motor function, attention, and visuospatial performance in these children showed decrements associated with hair mercury concentrations.100 Patients in an internal medicine practice in San Francisco who consumed large fish regularly and had mercury blood levels above 5 mcg/L presented most commonly with fatigue, hair loss, trouble thinking, memory loss, muscle aches, and headaches.101 One peculiar symptom in this group was a metallic taste in the mouth.
Mental health symptoms are also quite common with mercury toxicity. Evidence linking mercury exposure to psychological disorders has been accumulating for the last 60 years. The recognized psychological symptoms of mercury include irritability, excitability, temper outburst, quarreling, fearfulness, restlessness, depression and, in some cases, insomnia. In a study of individuals with amalgam filling who had them removed, the majority noted psychological improvements. The greatest improvements were found in anger outbursts, depression, irritability, and fatigue.102 None of these manifestations were too surprising when related to the effect of mercury on reducing the serotonin effect. The association of mercury to depression has stimulated some interesting questions as to whether mercury toxicity was to blame for Sir Isaac Newton’s health problems of 1692 to 1693.103
The most common presentations of toxic damage to the endocrine system are:
1. Sleep disturbances or changes in energy level or mood.
2. Alterations in weight, appetite, and bowel function.
3. Sexual interest and function change; in females, any menstrual change.
4. Changes in temperature perception, sweating, or flushing.
The most common endocrine diagnoses associated with xenobiotic burden include:
Pesticides, solvents, aromatic hydrocarbons, heavy metals, and chemical dusts have been associated with both male and female infertility.104
Herbicides and Organophosphate Pesticides
A study of Ontario farmers showed an association of female pesticide use with fecundity,105 and the relative risk of being infertile from pesticide exposure was 3.8. Five compounds reduced females’ fecundity rates 24% to 49%: Dicamba (0.51), glyphosate (0.61), 2,4-D (0.71), organophosphates (0.75), and thiocarbamates (0.76). Glyphosate is commonly known as “Roundup” and is highly advertised in print and on TV for its safety, yet was clearly associated with infertility in this study. In a study of Spanish greenhouse sprayers exposed to OPs, higher spontaneous abortion rates were found, along with higher rates of depression and headaches.106 Although these health problems were clearly evident, no significant decrease in erythrocyte acetylcholinesterase was found. This reproduced other studies showing apparent adverse organophosphate health effects without the expected serum finding indicating an acute OP toxicity. A study in California farming counties showed a clear association with pesticide spraying and fetal death due to congenital anomalies.107 In this study, when an OP or carbamate pesticide was sprayed in one of eight adjacent square miles of women’s residences during the third to eighth week of pregnancy, the OR of fetal death was 1.4. When the spraying occurred within one square mile of a residence, the OR increased to 2.2.