Medical Hypotheses 16: 397-402, 1985

FLUOHIDE HYPHHSNNSITIVITY IN mAINS TAP WiirLB D.NNONST~kD BY

SKIN POTENTIAL CHANGES IN GUINEA - PIGS

A. Lewis and C.W.M. Wilson, Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, end Division of Geriatric Medicine, Law Hospital, Carluke, Lanarkshire.

Changes in skin electric potential following immune challenge provide a sensitive measure of the intensity of the allergic challenge. In guinea-pigs sensitised to Sodium Fluoride, Fluoridated keins Tap Water, or Ovalbumen with dodium Fluoride, and then challenged with one part in one million Sodium Fluoride, significant allergic responses were produced. Cvalbumen alone caused an allergic response only after a second challenge. Purified Water did not result in any immune response. It is concluded that Fluoridated Mains Tap Water is capable of causing an allergic response similar to that produced by Sodium Fluoride alone, and that Cvalbunmn potentiates the immune response to Sodium Fluoride.

INTkODUCl!ION

Change in tissue electric potential has been used to demonstrate antigen/antibody reactions in atopic individuals after challenge with specific allergens (8, 10, 13). The diagnostic value of this test has been confirmed in guinea-pigs by Lewis and Wilson (2, 3). In the present in- vestigation, this method for detection of antigen-induced reactions has been used to investigate the responses to sodium fluoride in guinea-pigs previously sensitized to fluoridated Irish domestic mains tap water, or to sodium fluoride by itself or in combination with ovalbumsn. Irish domestic mains tap water contains fluoride at a concentration of one part in one million. The experiment was also designed in order to examine the effect of ovalbumen on the fluoride-induced reactions.

AUTHOD

A standard solution of sodium fluoride was prepared from pure sodium fluoride powder (Water Technology Ltd., Cork, Ireland) containing 2.21 mg/L in purified water (Hiogen Water, Water Technology Ltd., Cork, Ireland).

T his solution contai Crystalline ov HDH, Poole, England f

ed 1.0 mg/L fluoride ions. was prepared at a concentration of 10.0

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sodium fluoride solution. Pure ovalbumen solution was also prepared in Biogen water at a concentration of 10.0 mg/L. Dublin domestic mains tap water was obtained from the Dublin lvlains Tap Water supply from ~allymore and Vartry reservoirs. During the period January-December 1979, the con- centration of fluoride in this water varied from 0.950 to 1.050 parts per million. Purified Biogen water was used as the control water.

Ten Male Duncan-Hartley guinea-pigs weighing 400 - 500 gms were used as experimental animals. They were fed on laboratory pellets for seven days. They had free access to Dublin domestic mains tap water from con- tainers in the cages. L-Ascorbic acid was added to this water at a con- centration of 50 mgf100 mls in order to stabilise body potential (3). The animals were divided into five groups of two animals which were housed in separate cages. On the 8th day (Day 0), they received treatments as shown in Table 1.

TABId 1

Sensitising intraperitoneal injections made up in 0.5 ml Biogen Purified Later administered on Day 0.

GROUP TREATEENT RKEIWD

A 0.5 ml Biogen water B 0.5 ml NaF ;jolution 2.21 mg/L C 0.5 ml Tap water D 0.5 ml OA 10.0 mg/L + haF 2.21 mg/L (1 mg/L Fluoride) E 0.5 ml OA Solution 10.0 mg/L

Twenty four hours and twenty five days after the initial sensitising injections, Groups B - E were given intraperitoneal challenge doses of 0.5 ml fluoride solution. Group A acted as control group and received sensitising and two challenge injections of Biogen water. Skin potentials were recorded 5 minutes before, previously described (3).

and 6 and 10 minutes after challenge, as Skin potentials were recorded from two pairs of

points on the back of each guinea-pig, one of each pair being used for the reference, and one for the probe electrode. Four measurements were taken from each animal at each recording. Results are expressed as means and standard errors for each group. The two AgCl electrodes were connected to the leads of a high-input DE-2 Sinclair millivoltmeter. During the periods of measurement of the potentials, they invariably reached a stable level within one minute.

RESULTS

Table 2 shows the skin potential values which were recorded after the first and second challenge injections in the five groups.

ho significant changes occurred in skin potential following the challenge injections of Biogen water in the control group. Sodium fluoride made up in Biogen water caused a highly significant reduction in skin potential 6 and 10 minutes after the first and second challenge injections. This indicates that a challenge dose of sodium fluoride is capable of

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causing a profound immune responf~e, both within 24 hours and 25 bye after sensitization. After the first challenge dose of sodium fluoride solution to the group eensitized with tap water, a silprificant immune response occurred. The first response was less severe than that which occurred after the second challenge dose of sodium fluoride solution 25 deya later. The first challenge dose of sodium fluoride administered to the group which had been sensitized with ovalbuaen in sodium fluoride solution, caused a sig- nificant fall in akin potentials 6 and 10 minute@ later. After adminiatra- tion of the second challenge dose to these animals, larger and more highly significant reductions in shin potentials were recorded. Guinea-pig8 which were eenaitized with ovalbmn alone did not show any appreciable change in skin potentials after the first challenge injections of sodium fluoride. After the second challenge dose of sodium fluoride 25 daya after the sensitizing injections of ovalbumen, a reduction in skin potential was recorded. However this reduction was not statistically significant.

TABLE 2

Skin electric potentials preceding, and 6 and 10 minute8 after, challenge intraperitoneal injections of 0.5 ml sodium fluoride (1 mg/L fluoride), administered 24 hours and 25 days after sensitization. llsans and SE, P= 8.

After 24 hours challenge

Group ORliIlS 6mins 10 mins

A (Biogen Water) 5.5 5.0 1.84 4.5 1.11 B (hap)

+ 1.36 -+ + 5.75 + 1.43 1.0 0.18 -+ 1.0 0.16

(P<O.O05) 2

C (Tap Water) (P<O.O05)

5.0 + 1.33 2.5 1.30 +

D(OA+NaF) (P<O.O5)

3.0 21.2

5.0 L 1.30 1.0 0.12 + 2.0 1.0 +

E (On)

(P<O.O05) (P(O.05)

5.75 -+ 1.23 6.0 1.53 2 5.5 1.80 +

After 25 days challenge

A (Biogen hater) 4.75 B (NaPI

+ 1.23 4.50 1.86 + 5.0 2.08 + 5.75 + 1.45 1.50 1.12 +

(P<O.O25) 1.0 + 0.15

C (Tap Water) (P(O.025)

4.25 + 1.75 1.50 + 0.96 (P<OTO5)

1.50 0.85 +

D(OA+NaF) (P<O.O5)

8.75 + 2.86 1.0 0.12

E (Oa)

(P<O.O05) + 1.0 0.85 + (PYO.0025)

8.0 22.7 4.5 21.3 7.0 2 2.1

P values in comparison with 0 time.

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CONCLUSION

It has been shown that the standard procedure for production of an antigen-antibody reaction by injection of a challenge dose of ovalbwnen into ovalbumen-sensitized guinea-pigs caused a significent reduction in skin electric potential, in comparison with the absence of any such change in similarly sensitized guinea-pig8 to whom a challenge dose of saline was administered (3). Thia procedure provide8 a rapid, effective, and sensitive method for recognising and measuring the occurrence and intensity of tha antigen-antibody reaction in specifically sensitized guinea-pigs. The antigen induced change in electric potential can be detected at a point distant from the challenge site. In the present investigation, the adult guinea-pigs, like the Irish human population since 1954, had been receiving fluoridated mains domestic tap water from the time of birth. The guinea- pigs, in these circumstances, like humans when subjected to continuing allergenic challenge, had the opportunity to respond immunologically to the potential sensitising agent in a specific fashion, if the fluoride was capable of acting in this way.

The present results demonstrate that sodium fluoride, made up in purified water at a concentration similar to that in Irish domestic main8 tap water, caused a significant immune response in sensitized guinea-pigs, as demonstrated by a significant reduction in skin electric potential, both 24 hours and 25 days after administration of challenge doses of the sodiru fluoride. This indicates that the sodium fluoride was acting as an immune sensitizing agent. The significant reduction in electric potential demon- strated that antibodies were present in the back skin at a site distant from the point of administration of the initial sensitizing, and subsequent challenge, injections of sodium fluoride. In rats ovalbumen antibodies are present in the serum for only 5 - 6 days following the sensitizing dose of ovalbumen (4), after which they have been excreted or become tissue- bound. The highly significant reduction in electric potential response to the first challenge injection of fluoride therefore suggest8 that this injection could have acted as a challenge in guinea-pigs which already had fluoride antibodies in their skin as a consequence of oral sensitization to fluoride. This suggestion is further supported by occurrence of a highly significant fall in electric potential after the challenge dose of ovalbumen with fluoride, and the absence of any response to a challenge dose of ovalbumen by itself. The intense response to the second challenge dose of fluoride by itself occurred after the fluoride antibodies to the first challenge dose of fluoride has become tissue bound. The absence of a similar immune response in the control animals receiving biogen water in place of sodi.um fluoride confirms the specificity of the immune response to fluoride.

Irish tap water contains residual chlorine, chloride ions, ammonia in free and bound forms, nitrites, nitrates, sulphate and aluminium, in addition to fluoride. The observation that the challenge dose of sodium fluoride produced a significant reduction in skin electric potential, after the guinea-pigs had been previously, specifically sensitized with tap water, indicates that the fluoride in Irish domestic mains tap water is capable of producing an immune response under controlled experimental conditions.

This response was maintained for 25 days after the initial sensitizing

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injections.

When the challenge doses of sodium fluoride were injected into the _gea-pigs sensitized with ovalbmn, no significant changre ocau?rd in skin potential. When however ovalbumsn was used in combination with sodium fluoride as the sensitizing agent, a massive and highly significant reduction occurred in skin potential following challenge with sodium fluoride 24 hours later. The immune response to this challenging egent was consiclerably greater 25 day8 later. It iias also greater thsn the immune response nhen sodium fluoride alone was the sensitizing agent. This demonstrates that the ovalbumen significantly potentiates the immune res- ponse to fluoride. It is possible that the fluoride, in virtue of the fact that sensitization to this molecule had already occurred, uas acting as a hapten, and that this enabled sensitization to the ovalbumsn to t&s place at the time of its first administration. Alternatively, it is possible that the combination of the ovalbumen with the fluoride activated the non-opecific pathway which operates through lysine residues (1). Throughthio pathway, the fluoride might activate a more massive response than the ovalbwn alone, when activating a specific antibody response.

The results confirm the value of the procedure for measuring change in tissue electric potential in order to assess the specificity and severity of immune responses. They have demonstrated that the fluoride present in domestic tap water can act as a potent aller tigations have demonstrated that toxicity (6 P

n by itself. Earlier inves- and immune sensitivity (7) to

fluoride occurs in humans. The present results provide definite confirma- tory evidence that fluoride immune sensitivity is produced by administration of fluoridated tap water. It has been shown that hypersensitivity to fluoridated Irish domestic mains tap water occurs in children and adults (11, 12). The allergenic bypersensitizing action of this water wae con- firmed by the appearance of characteristic allergic symptoms in the patients when challenged with this water, by the observation that exclusion of the patients from the challenge antigen& water, as in the case of other challenging allergens, resulted in disappearance of patient symptoms, and by the fact that the symptoms could be effectively treated and prevented by specific neutralisation against the mains water. It is possible that other contaminants than fluoride in domestic mains water are capable of producing immune responses (5). However the specificity of the sensitization and challenge in the present laboratory investigation has demonstrated that the fluoride ion, present in the tap water, is definitely responsible for the development of sn immune response in sensitized individuals.

Yhen administered in combination with ovalbumen, ths immune respon8e to fluoride is significantly enhanced. This finding demon&rake that food protein potentiatea the immnne response to fluoride. Attention has been drawn elsewhere to this capacity for one allergen to potentiate the immune response to another allergen during a period of challenge (9). The potentiating effect of food protein on the immune response to fluoride w account for the earlier difficulties in demonstrating specific fluoride sensitivity in humens. The specific fluoride sensitivity may have been masked by simultaneous potentiation of the patients' allergic reactions to food proteins. It is noteworthy that all the patients shown to be sensi- tive to water contaminants were also found to be sensitive to foods snd extrinsic antigens (11, 12). This observation suggesto that in humens, as

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in guinea-pigs, food and water contaminant sensitivities tend to occur in the same individuals and msy be potentiating. Since all food is contained in, and cooked and prepared with, mains tap water, it is possible that water contaminants produce a uore continuous and eerious immune challenge to humans than individual foods.

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