Fluoride Toxicity Research Articles

Fluoride Toxicity In Domestic Animals Of Chandrapur District, Maharashtra, India

Fluoride Toxicity In Domestic Animals Of Chandrapur District, Maharashtra, India

Fluoride Toxicity from Oral Medicament

Fluoride Toxicity from Oral Medicament

A Brief and Critical Review of Chronic Fluoride Poisoning (Fluorosis) in Domesticated Water Buffaloes (Bubalus bubalis) in India: Focus on its Impact on Rural Economy

In the rural areas of India, fluoridated drinking water, industrial fluoride pollution and fluoride-rich feed phosphate supplements are the major sources of fluoride exposure for domesticated water buffaloes (Bubalus bubalis). However, the fluoridated drinking groundwater is the commonest and principal source of fluoride exposure for these ruminants. Chronic fluoride exposure for long-time deteriorate the animal health and causes the fluorosis disease. In the country, the drinking groundwater sources of villages are contaminated with fluoride beyond the threshold value, 1.0 or 1.5 mg/L. At 1.5-1.7 ppm fluoride concentration in drinking water, 62.2% and 21.6% calves and 55.9% and 48.3% adult buffaloes are found to be afflicted with dental and skeletal fluorosis, respectively. The maximum prevalence of dental and skeletal fluorosis, 96.8% and 34.4% in calves and 66.9% and 60.2% in adult buffaloes has also been reported at or > 3.0 ppm fluoride in drinking water, respectively. However, calves are found to be more sensitive and susceptible to chronic fluoride toxicity or fluorosis. Besides the deterioration of the animal health, chronic fluoride intoxication in buffaloes also weakens the rural economy which has been focussed in the present review. In this communication, besides the status of chronic fluoride intoxication in the form of osteo-dental and non-skeletal fluorosis in buffaloes, determinations, bio-indicators and bio-markers of fluorosis and prevention and control of chronic fluoride poisoning in these animals are also considered and briefly and critically reviewed. Simultaneously, research gaps are also identified and highlighted them for further research study on chronic fluoride intoxication in buffaloes. Findings of this review may help in preparation of health policy for improving the health and mitigation of fluorosis in economically important buffalo animals in the country.

Sodium fluoride induce alterations in glycogen metabolism in freshwater catfish, Clarias batrachus (Linn.)

Effect of fluoride toxicity on glycogen metabolism in fresh water catfish Clarias batrachus was evaluated after their exposure to two sublethal concentrations of fluoride (35 mg F ion/L and 70 mg F ion/L) for 60 days and 90 days. Alterations in glycogen content in muscle, liver, and testis tissues were recorded. The insignificant decreased level of glycogen content was recorded in muscle and liver tissues at lower concentration of F in both durations. However its elevated level was recorded at higher concentration of F in both tissues at both durations. The duration and concentration dependent depleted glycogen level was recorded significant (P

Fluoride supplementation during pregnancy does not reduce caries in offspring

Fluoride represents an essential element in dental development and safety. Fluoride deficiency is associated with dental cavities. The main dietary source of fluoride is in water, followed by fluoridated toothpaste, tea, seafood, canned sardines or shells as well as medicinal supplements. The adequate intake ranges from 0.7 mg/day for toddlers, to 3 mg/day for adult females and 4 mg/day for adult males. The fluoride intake can present a difficulty in assessment, especially during pregnancy, so the question remains: is fluoride supplementation recommended during pregnancy to prevent dental cavities in the offspring? The correct answer is ‘No’. Fluoride supplementation during pregnancy does not reduce the incidence of caries in offspring. Centers for Disease Control and Prevention (CDC) does not recommend fluoride supplementation due to its action mechanism; fluoride has beneficial effects only after the tooth eruption takes place through a topical mechanism. Fluorosis (fluoride toxicity) in offspring has not been described secondary to excessive ingestion of fluoride of the mother during pregnancy, but there are studies correlating neurotoxic effects in the infants from mothers who received fluoride supplementation.

Fluoride sensitivity in freshwater snail, Bellamya bengalensis (Lamarck, 1882): An integrative biomarker response assessment of behavioral indices, oxygen consumption, haemocyte and tissue protein levels under environmentally relevant exposure concentrations

There is limited information on fluoride toxicity and risk overview on ecotoxicological risks to aquatic invertebrate populations particularly molluscan taxa. This necessitated the assessment of toxicity responses in the freshwater snail, Bellamya bengalensis exposed to environmentally relevant concentrations of sodium fluoride. Under lethal exposures (150, 200, 250, 300, 400 and 450 mg/l), the median lethal concentrations (LC50) were determined to be 422.36, 347.10, 333.33 and 273.24 mg/l for B. bengalensis at 24, 48, 72 and 96 h respectively. The rate of mortality of the snails was increased significantly with elevated concentrations of the toxicant. The magnitude of toxicity i.e., toxicity factor at different time scale was also higher with increased exposure duration. Altered behavioural changes i.e., crawling movement, tentacle movement, clumping tendency, touch reflex and mucous secretion in exposed snail with elevated concentrations and exposure duration. Similarly, oxygen consumption rate of the treated snail also lowered significantly during 72 and 96 h of exposure. Under 30-day chronic exposures (Control-0.00 mg/L; T1–27.324 mg/L; T2–54.648 mg/L), protein concentrations in gonad and hepatopancreas of exposure groups was significantly lowered. Chronic exposures also revealed lowered haemocytes counts in exposure groups. The potential for loss of coordination, respiratory distress and physiological disruption in organisms exposed to environmentally relevant concentrations of fluoride was demonstrated by this study. The estimation and magnitude of toxicity responses are necessary for a more accurate estimation of ecological risks to molluscan taxa and invertebrate populations under acute and chronic fluoride exposures in the wild.

Health risk assessment of nitrate and fluoride toxicity in groundwater contamination in the semi-arid area of Medchal, South India

Hydrogeochemical controlling variables for the high rate of groundwater contamination in a shallow hard rock aquifer of the semi-arid region of Medchal District, Telangana State, South India, and its associated health risk to children and adults were studied in detail. A total of 56 groundwater samples were analysed for major ion chemistry in pre- and post-monsoon seasons in 2019 year. Spatial distribution, hydrochemical facies, water–rock interaction, health risk assessment, carcinogenic risk, and principal component analyses were carried out to assess the water quality. Spatial distribution of nitrate and fluoride concentrations, high values were observed in the northern, southern, central, and south-western parts of the region. In terms of NO3−, about 88% and 63% and for F− 45% and 32% of the groundwater samples are non-acceptable limits of nitrate 45 mg/l and fluoride 1.5 mg/l during pre- and post-monsoon seasons, respectively. Modified Gibb’s plot showed the majority of the area is dominated by rock dominance and evaporation mechanisms. Statistical analysis reveals that water chemistry is governed by weathering of feldspar minerals and the cation exchange reaction mechanism. The petro-graphical studies revealed the dominance of plagioclase, orthoclase, quartz, biotite, apatite, and hornblende minerals in the host rock. The factor analysis results reveal that the geogenic and anthropogenic activities contribute to groundwater chemistry. Health risk assessment was carried out by calculating the hazard quotient (HQ) on the basis of intake exposure of groundwater, as per the USEPA. Results were obtained for total hazard index value greater than 1 for adults and children, which causes non-cancerous health effects.

Fluoride Toxicity from Oral Medicament

Fluoride Toxicity from Oral Medicament

Therapeutic effect of taurine on sodium fluoride toxicity in chicks

The study aimed to investigate if taurine could help reduce sodium fluoride-induced toxicity in chicks. The chicks in this study were divided into four equal groups, each with eight chicks: the control group, sodium fluoride 20 mg/kg group, taurine 3 g/kg group, the fourth group was dosed with each of the sodium fluoride 20 mg/kg and taurine 3 g/kg groups all groups were dosed orally. The dosing was set at 5 days/week for 4 weeks. After 2 and 4 weeks of treatment, the group treated with taurine alone or with sodium fluoride showed an improvement in neurobehavioral and motor activity, as evidenced by a reduction in the duration of chick immobility in the immobility test and an increase in the number of squares cross in the open field test compared to the group treated with sodium fluoride alone. The level of ALT enzyme and calcium in the group treated with sodium fluoride increased significantly compared to the control and taurine group alone, and with sodium fluoride, AST and creatinine levels increased significantly after 4 weeks of sodium fluoride treatment compared to the other groups. When it came to measuring malondialdehyde and glutathione, the sodium fluoride group alone showed a significant increase in malondialdehyde and significant decrease in glutathione after 2 and 4 weeks of treatment when compared to the control and the other groups. The histopathological examination confirmed the previous findings, with the histological sections of the liver, kidney, and brain showing a significant improvement in the group treated with sodium fluoride and taurine after four weeks of treatment. We conclude from this study that taurine has a clear therapeutic effect against oxidative stress, as evidenced by behavioral and motor behavioral effects, as well as levels of glutathione, malondialdehyde, and liver function enzymes, and serum creatinine, as well as histopathological examination of the brain, kidneys, and liver.

Significance of Inflammation and Apoptosis in Hepatocellular Death in Rat, Co-treated with Arsenic and Fluoride.

Health effects elicited by combined environmental exposures to xenobiotics, in many instances, still remain unresolved. One of these examples is the combined toxicity of arsenic and fluoride. The present study was undertaken to delineate the role of inflammation and apoptosis in hepatocellular death caused by co-exposure to arsenic and fluoride in rat. Sodium arsenate (4mg/kg body weight) and sodium fluoride (4mg/kg body weight) were administered to female Wistar rats, individually and in combination, for 90days. Results on tumor necrotic factor-α (TNF-α), interleukin-12 (IL-12), and comet assay showed increased values in comparison to those obtained in arsenic- or fluoride-treated rats. Results on NO, TBARS, and caspase-9 showed higher values than fluoride-treated rats but lower levels than arsenic-treated rats. It is hypothesized that increased generation of nitric oxide induces the release of cytokines that activates caspase-9. Caspase-9 promotes the synthesis of caspase-3 that executes apoptosis. Histopathological observations on apoptotic bodies and Kupffer cells support these observations.

Effect of boric acid on sodium fluoride toxicity in chicks

The aim of this study to explore the therapeutic effect of boric acid on the neurobehavioral (motor activity) level, and histopathologic changes in the brain, liver and kidneys against fluorosis. In this study rose chicks have been used and determined medium lethal sodium fluoride dose at 346.5 mg/kg orally. The chicks divided into four random groups each one consists of 10 chicks. The first group considered to be a control group, the second received 20 mg/kg of sodium fluoride, the third group received 10 mg/kg of boric acid and the fourth received 20 mg/kg of sodium fluoride and boric acid at the same previous dosages. After two weeks of daily treatment, neurobehavioral measures were taken, the use of boric acid has a major effect to improve the neurobehavioral measurement and develop complications of ALT, AST, creatinine, Ca, MDA. The results indicate that boric acid may be a therapeutic agent against the fluoride toxicity of the brain, liver and kidney. This result support by histopathological changes which represented by inflammation, congestion of portal vein and dilation of sinusoids in the liver and vacuolation, vasogenic edema and gliosis in the brain and Kidney of showed segmentation of glomeruli, dilation of Bowman’s space, necrosis of epithelial cells renal tubules and hemorrhage of NaF group, while the liver of the NaF with boric acid group showed an improvement the results of histopathological examination of the liver, brain and kidneys compared to the NaF group alone. The results revealed that boric acid has a preventing effects against fluoride after two weeks of treatment with boric acid.

Germination and morphological responses of Triticum aestivum L. to different concentrations of fluoride

A laboratory experiment was conducted on germination papers to study the effect of fluoride (F) at 0 (T1), 50 (T2), 100 (T3), 200 (T4), 250 (T5) and 300 (T6) ppm on germination and morphological parameters in wheat (Triticum aestivum L.) variety, HUW-234 at 2, 4 and 6 days after initiation of germination process. Fluoride toxicity caused reduction in germination per cent, germination index, coefficient of velocity of germination and germination energy (%) while mean germination time increased with fluoride concentration. Root and shoot lengths and dry matters decreased with increased concentrations of fluoride. Ratio of root: shoot weight increased with increased concentration of fluoride. Elongation of seminal roots was adversely affected by increased fluoride level. Increased fluoride level in the germination medium decreased RGR of seedlings progressively. Present study revealed that enhanced fluoride concentration in germination medium caused reduction in germination and germination related parameters.

Effect of Fluoride Toxicity on Some Haematological Indices in Human Population

The present study aimed to determine the toxicity of Fluoride on haematological indices in some population residing in fluoride endemic area of Dongergaon village of Chandrapur district, Maharashtra, India. The study group was selected on the basis of elevated level of serum fluoride and symptoms of skeletal fluorosis. Various haematological parameters such as Hb, TEC, TLC, HCT and PLT count were analysed. Students t-test was applied for statistical analysis and the result showed that values of haematological indices in study group were significantly declined as compared to control group. The outcomes of the present study indicated the reduction in Hb, TEC, HCT, increase in TLC and non-significant change in PLT.

Assessment of renal and hepatic dysfunction by co-exposure to toxic metals (Cd, Pb) and fluoride in people living nearby an industrial zone

Togo's phosphate processing plant at Kpeme discharges waste, containing Cd, Pb, and fluoride, into the sea and on the soil. Heavy metals toxicity on kidneys and the liver has been studied. However, fluoride toxicity on these organs remains to be investigated. The present study deals with the variation in renal and hepatic functioning parameters due to fluoride, Cd and Pb. Totally, 350 volunteers were recruited from five different localities around this phosphate processing plant for sample collection. Cd and Pb contents in blood samples were determined by spectrophotometry and fluoride by the titanium chloride method. Biochemical parameters were measured using Biolab kits. The pollutant contents were elevated in polluted areas where ASAT, ALAT, creatinine, and urea increased, and total protein decreased. Correlation and multivariate tests showed that fluoride is related to the various pathologies mentioned. PCA revealed that phosphate processing in Togo is a source of renal and hepatic toxicity.

Fluoride Toxicity Limit—Can the Element Exert a Positive Effect on Plants?

The problem of fluoride toxicity to living organisms is the subject of many studies. Its effect, not always toxic, on the human organism has been well documented. However, although the phytotoxicity of the element has been proved, this issue is still being investigated. It seems to be still relevant due to the progressive pollution of the environment and fluoridation of water. Assuming that the source of food for humans is plants, the content of fluoride in fruits and vegetables is important for human health. In the available literature, fluoride has been demonstrated to be phytotoxic at the level of cell transformations, biometric plant parameters, development of resistance, and biochemical processes in plants. However, several studies have provided information on improvement of certain plant parameters, e.g., the length of roots or shoots, caused by low fluoride doses and improvement of respiratory indices. The aim of this study was to analyze changes caused in plants by exposure to fluoride and to determine its beneficial effects based on the latest literature reports. It was based on the latest knowledge from the last 8 years. Attempts were made to compare earlier research results with contemporary items. In conclusion, the analysis has shown that, although some sources provide information on the positive effect of small fluoride doses, the impact of this element requires further investigations, as has not been fully elucidated.

Molecular Mechanism of Changes in Gene Expression of Cytosolic Cu/Zn SOD, Lipid Peroxidation and Hepatic Function Impairments during Experimental Fluorosis

Background: Long term intake of high amount of fluoride leads to fluorosis causing metabolic, functional and structural damages affecting many tissues and organs including dental and skeletal manifestations. The liver is the most susceptible organ to fluoride toxicity because of its active and major role in digestion and detoxification.
 Aim: The present study aims to elucidate the effects of sodium fluoride on hepatic function biomarkers, lipid peroxidation and gene expression of Cu/Zn SOD in albino rats.
 Materials and Methods: Wistar albino rats were randomly assigned to three groups. The control rats were given 1 ml deionized water orally for 40 days. Groups II and III were administered 300 and 600 mg NaF/kg b.w. /day for the same period. Animals were sacrificed under anaesthesia, liver tissue was excised and used for biochemical and molecular analysis. The level of fluoride and lipid peroxidation (MDA) as well as reduced glutathione (GSH) content was determined. The activities of cytosolic copper/zinc superoxide dismutase (Cu/Zn SOD), aminotransferases (ALT and AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) in the hepatic tissue were determined. The analysis of gene expression of Cu/Zn SOD in the liver was determined using Real-time PCR.
 Results: The results revealed significantly (P<0.0001) higher concentration of fluoride and MDA in the liver of rats exposed to fluoride when compared to control. The GSH content reduced significantly (P<0.0001) in fluorotic rats. The activities of hepatic function biomarkers viz; ALT, AST, LDH and ALP elevated significantly (P<0.0001) compared to the control. An elevation of 108.60% (ALP), 121.45% (AST), 33.77% (LDH) and least 24.40% (ALT) was found in rats treated with 300 mg/L fluoride and maximum elevation of 226.20% (ALP), 211.52% (AST), 57.75% (LDH) and least 56.79% (ALT) was registered in rats exposed to 600 mg/L fluoride in drinking water. The activity of cytosolic Cu/Zn SOD decreased significantly (P<0.0001) in fluorotic rats. Pearson’s bivariate correlation and simple linear regression analysis exhibited strong positive correlation between level of hepatic tissue fluoride and activities of ALT (Pearson r= 0.85), AST (Pearson r= 0.94), LDH (Pearson r= 0.89), ALP (Pearson r= 0.86) and in MDA (Pearson r=0.984) while negative correlations existed between levels of fluoride and GSH (Pearson r=-0.93) as well as activity of Cu/Zn SOD (Pearson r= -0.99). The gene expression of cytosolic Cu/Zn SOD was significantly (P<0.0001) reduced in fluorotic rats.
 Conclusion: The present study revealed that fluoride declined the antioxidant activity of hepatic Cu/Zn SOD at biochemical as well as molecular level which leads to oxidative stress and tissue damage. This further affirms by increased activities of hepatic function biomarkers in correlation with high fluoride level during experimental fluorosis.

Effects of Tamarindus indica Fruit Pulp Powder on Blood and Serum Profile of Fluorosed Rats

Aims: Water containing a fluoride concentration of up to 1.0 mg/L is safe. It was found that the level of fluoride in groundwater is higher than surface water, which may lead to various health problems related to fluorosis. T. indica fruit pulp powder may help to reduce fluorosis condition in rats.
 Methods: In the present study, healthy adult rats of Wister strain (Rattus norvegicus) weighing between 180-200gm were used for experiments. The animal was divided into five groups. Group I, control rats received only tap water (0.9ppm F). Group II rats were treated with Tamarindus indica fruit pulp powder (4mg/day/rat) for 60 days. Group III rats were exposed to Fluoride water (100ppm F) for 60 days. Group IV treated with fluoride water (100ppm F) along with T. indica fruit pulp powder (4mg/day/rat) for 60 days. whereas, group V rats were ingested fluoride water (100ppm F) for 60 days and Withdrawal from treatment for 30 days. After respective treatment animals were autopsied and biochemical parameters of blood (erythrocyte and leukocyte count, hemoglobin, hematocrits); serum (protein, glucose, cholesterol); serum enzyme activity of SGOT SGPT, Acid phosphatase, and serum fluoride and calcium level was estimated using standard techniques.
 Results: Results revealed that total erythrocyte, hemoglobin, and hematocrit values were (P=.01) reduced significantly after rats were exposed to fluoride (100 ppm) water for 60 days. whereas total leucocyte count increased with fluoride (100ppm) water exposure for 60 days as compared to control values. The serum enzyme activity of SGOT, SGPT, and alkaline phosphate elevated significantly (P=.01), whereas enzyme activity of acid phosphatase diminished following fluoride water treatment to rats, as composed to control value. The level of serum fluoride enhanced significantly (P=.01) in fluorotic rats as compared to the control value. However, when fluorosed rats were treated with T. indica for 60 days restored all altered parameters almost to control value. Withdrawal of fluoride water for 30 days revealed that there is partial recovery in all parameters studied.
 Conclusion: Fluoride water consumption increases free radical generation, limits enzyme activity, and results in altered hematological and blood biochemistry. Tamarindus indica pulp powder was found to be beneficial in mitigating fluoride toxicity in rats.

Impact of F80M and F83M mutations on the functionality of fluoride ion channel elucidated in microsecond level molecular dynamic simulation

Fluoride ion channels of the Fluc family plays a critically important role in combating environmental fluoride toxicity. As per the crystal structure of these fluoride ion channels, the pore region is densely packed with a series of hydrogen bond donating residues arranged in a ladder fashion creating an ion conducting pathway. In earlier studies, it was revealed that although the ion conducting pathway polarity is highly conserved, however the functionality of the channel protein depends on several residues at particular positions. While, a threonine at end of the pore is critically important in forming initial interactions, two phenylalanines at the central region coordinate F- transportation through the channel. It was also revealed that these two phenylalanines cannot be substituted by any other aromatic, polar or non-polar residues without hindering the functionality with exception of methionine. In another study, it was revealed that these two phenylalanines F80 and F83 when mutated with methionine; F80M lead to active state, while the F83M has lead to inactivity of F- anion conductivity. However, the exact atomic level detailing on how exactly these mutations have impacted the conductivity remained elusive. In this scenario, in this present study, we have modeled these two mutations and performed a microsecond level simulation on each mutation compared with wild type towards understanding the atomic level detailing revealing several insights on what exactly happening at these residues responsible for the selective conductivity of F- ions. Communicated by Ramaswamy H. Sarma

Prediction on the combined toxicities of stimulation-only and inhibition-only contaminants using improved inverse distance weighted interpolation

The evaluation of ecological risks of contaminant mixtures to organisms is very challenging due to the non-linear response of organisms to each component, especially under the co-existence of both stimulators and inhibitors. Whether the stimulatory effect can reduce or even offset the inhibitory effect would be critical to the risk assessment and the treatment measures of mixed pollutants. Here, the combined toxicity of sodium fluoride (NaF), a stimulator with stimulation rate >100%, and six compounds that cannot induce hormesis (four ionic liquids (ILs) and two pesticides) were studied. The time-dependent toxicity of each toxicant on Vibrio qinghaiensis sp.-Q67 was investigated at 0.25, 2, 4, 6, 8, 10 and 12 h. Results showed that four ILs and two pesticides failed to induce hormesis, while NaF induced hormesis from 2 to 6 h and induced stimulation only after 6 h and reached its maximum (650%) at 12 h. All mixture rays with NaF induced hormesis at different times. In the four NaF-IL mixture systems, the absolute value of maximum stimulation demonstrated an upwards and then a downwards trend with the increasing of mixture ratio of IL. In two NaF-pesticide systems, the maximum stimulation effect declined with the increasing of the mixture ratio of pesticide. The toxicities of the mixture were successfully predicted by the improved inverse distance weighted interpolation, which are not able to be predicted by the commonly used concentration addition or independent action models. This paper shed lights on evaluating the hormesis of mixtures and the ecological risk of fluoride.

Estimation of Fluoride and Sirtuin1 in Patients with Diabetic Nephropathy in Kolar District of Karnataka, India.

Objective Prevalence of type-2 diabetes mellitus (DM) and diabetic nephropathy is growing rapidly in Asian countries, affecting low- and middle-income groups. One of the epidemiological issues of Kolar district is fluorosis; advanced glycation end product, carboxymethyl lysine (CML), and a molecule of interest Sirtuin1 are employed in the present study. In the correlation of fluoride with sirtuin1and CML with sirtuin1 of cases lies the important rationale of the study to assess the extent of kidney damage. Materials and Methods This is a comparative cross-sectional study with three groups, each with 70 patients, as follows: G1, control; G2, diabetes with diabetic nephropathy; and G3, type-2 DM without any complications. Informed written consent was obtained from all study patients. All the routine investigations were performed by fully automated Vitro 5, 1 Fs, Vitros. Fasting insulin was analyzed by Vitro eCI and glycated hemoglobin was estimated by BioRad D10. Sirtuin1, CML, and fructosamine were estimated by double antibody sandwich technique. Statistical Analysis The statistical analysis was performed by SPSS 20 (IBM) software. Means of normally distributed data were compared using analysis of variance (ANOVA), and not normally distributed data were compared by Kruskal–Wallis test. A p -value of less than 0.05 was considered statistically significant. Results A decrease in sirtuin1, serum, and urine fluoride of group 2 (34.74 [25.08–53.2], 0.24 [0.2–0.5], and 0.24 [0.16–0.41]) was observed compared with other groups. Increased CML and fluoride act as prooxidant, restricting the effect of sirtuin1 on cellular damage, causing further complications such as increased insulin resistance and decreased insulin sensitivity. Conclusion The alterations in serum sirtuin1 levels indicate the severity of damage due to stress during hyperglycemia and fluoride toxicity; hence, sirtuin1 can be considered as biomarker of aging. Subsequently, the correlation of CML, estimated glomerular filtration rate (eGFR), and fluoride with sirtuin1 indicates that increasing sirtuin1 may defend the forthcoming damage and could be considered in therapeutics.