EVIDENCE
AND MECHANISTIC APPROACH
OF THE PROTECTIVE EFFECTS OF HEAVY METAL
HIGH DILUTIONS IN RODENTS AND RENAL CELL CULTURES
Protective effect of metal high dilutions
A.DELBANCUT, M.P. BAROUILLET, J. CAMBAR
Groupe d'Etude de Physiologie et Physiopathologie
Rénales. Faculté de Pharmacie - 3, place de la Victoire - 33000, Bordeaux
(France).
1 - Introduction
2 - Personal results
3 - Mechanistic Approach of Heavy Metal High Dilution Protection
4 - Discussion and Conclusion
Chronic exposure to heavy metals (e.g cadmium or lead), which are major environmental toxicants, can be associated with a variety of pathologies such as renal, hepatic or nervous damages (Mery and Fillastre, 1983; Humes and Weinberg, 1986). For decades, several authors have attempted to reduce in vivo metal toxicity using chelating agents or other metals as competing agents. Another method successfully applied consisted of pre- or post-treatment using either low concentrations (Terhar et al.., 1965; Ito and Sawauchi, 1966; Yoshikawa, 1973 ) or high dilutions (Fisher et al.., 1987; Cazin et al.., 1987; Herkovitz and Perez-Coll, 1991) of the same toxic inducing agent. Critical rewiews on the effectiveness of serially dilute agitated compounds in experimental toxicology have been recently published (Linde et al., 1994; Cambar et al., 1994).
For twelve years, investigations have been carried out in our laboratory, aimed at underscoring and ascertaining the effectiveness of very high dilutions of heavy metals in experimental toxicology. Our previous in vivo experiments performed in rodents had clearly pointed out a fair protective effect of mercury high dilution in mercury-induced intoxication (Cal et al., 1986, 1988; Larue et al., 1985).
The recent development of cell cultures permitted to largely reduce animal experimentation and introduced a convincing alternative method for a better approach of molecular mechanisms at cellular level. To date, renal cell cultures can be considered as a fruitful tool for studying the nephrotoxic potency of numerous substances (Bruggeman et al., 1989; Lagroye et al., 1995; Toutain et al., 1995).
More recently in our laboratory, renal cell cultures were pretreated with very highly dilute cadmium or cisplatin prior to incubation with high cytotoxic concentrations of these metals. As a result, the high dilution pretreatment prevented the cytotoxic effect induced by high concentrations of cadmium or cisplatin; furthermore, these in vitro data could be correlated with our previously reported in vivo results. Consistently, a recent book reported similar in vitro results when other mammalian cell lines were post-treated with low doses of heavy metals (Van Wijk and Wiegant, 1994).
1.1 IN VIVO PROTECTIVE EFFECT OF HIGHLY DILUTE HEAVY METAL AS ASSESSED BY DEATH RATE
Female Swiss mice weighing 18-20 g were housed by 15 in a room where the lighting conditions (light/dark 12:12 with light on from 08.00 to 20.00) remained constant all year long. Food and water were available ad libitum. Two series of experiments were carried out to test the influence of highly dilute mercury pretreatment on HgCl2-induced mortality in mice and also to determine whether this effect is reproducible at different months in the year.
Low concentrations of mercury (10-18 to 10-30 M) were prepared by Dolisos laboratories (Paris, France) from a mother solution (1g Hg / ml in distilled water) by serial centesimal dilutions 1:99 and the solutions were succussed between the successive dilution steps.
After a standardization period of 10 days, the mice were daily injected i.p. throughout a 7-day period as follows: 0.5 ml of the 10-18 M and 10-30 M HgCl2 dilutions (pretreated groups) or agitated (succussed) distilled water (control groups). On the 7th day of treatment, one hour after the last preventive injection, the animals were given a single i.p. dose of 5, 6 or 7 mg Hg/kg of HgCl2.
Two hundred sixty five mice were pretreated with 10-30 M mercury according to the previous protocol with only 5 mg Hg/kg of HgCl2 at 5 different months in the year (october, december, february, april and june).
In all experiments, the mortality was recorded daily during ten days after intoxication to assess the kinetics of the mortality rate.
Statistical analysis was performed using both the frequency comparison test and the Cosinor method.
The results of these experiments clearly show that the pretreatment with mercury high dilutions can induce an important reduction of the letal toxicity of HgCl2, especially when the amount of HgCl2 administered is as close as possible to the letal dose LD50 value.
Indeed, as outlined in figure 1, when the mice received 5 mg/kg, 73.4% of the animals died in the control group, against 50% and 26.7% (p < 0,01) only in the samples pretreated with 10-18 or 10-30 M mercury respectively. Similarly, when the mice were given 6 mg Hg/kg, 78.5% of the controls died while 66.7% of the 10-18 M mercury-pretreated and only 35.7% of the 10-30 M mercury-pretreated mice were lost (p < 0,01). This protective effect disappeared when the highest challenging dose of Hg/kg (7 mg) was administered, since the mortality rates were 93.3 for the controls and 73.4 and 78.5% (NS) for the pretreated groups.
The experiments carried out all year long showed a constant replication of the protective effect brought about by 10-30 M mercury against 5 mg/kg induced-mortality.
Figure
2. Seasonal changes in
mercury high dilution protective effect against mercury induced mortality in
mice.
Figure 2 shows the difference in mortality rates between pretreated and control groups was 66.6% in October, 26.1% in December, 11.1% in February, 49.9% in April and 75% in June. Moreover, the effectiveness of the mercury high dilution against HgCl2-induced mortality exhibited a circannual rhythm (p < 0.01). The Cosinor analysis located the maximum of this protective effect in August.
1.2. IN VITRO
PROTECTIVE EFFECT OF HEAVY METAL HIGH DILUTIONS ASSESSED BY THE CYTOTOXICITY
RATE IN RENAL CELL CULTURES.
The protective effect of heavy metal high dilutions was assessed using an in vitro model applied to renal cell cultures. Cadmium or the vehicle was hundredfold serially diluted in the buffer (culture medium) and agitated for 30 s. We use very low cadmium concentrations (10-10 to 10-40 M).
Cultured cells issued from proximal tubular cell line (LLCPK 1) were incubated for 120 h with either dilute cadmium (pretreated cell group) or dilute culture medium (control cell group) and then intoxicated by massive amounts of cadmium. Cell viability was assessed by the neutral red test ; letality rate was compared to non-intoxicated cells. During the whole procedure (pretreatment and intoxication ), calf fetal serum (CFS) concentration was only 2 %, in order to prevent the chelation of the metal by proteins of the CFS. A protective index of very high dilution was determined by comparing the death ratio of the pretreated cell group with the control one. The first experiment is summarized in figure 3.
Figure
3. Protective effect of Cd
10-40 M
dilution against Cd intoxication in
tubular cultured cells.
Figure 3 shows LLCPK 1 cell mortality towards ponderal cadmium 24 h after intoxication, in a case where the cell pretreatment with either highly diluted solutions of cadmium (pretreated cell group) or diluted culture medium solutions (control cell group) lasted 120 h.
The difference between both cell groups was highly significant, fairly illustrating the protective effect of the 10-40 M cadmium dilution: cell mortality in the pretreated group was very low as compared with the untreated sample; 2.10-5 M cadmium induced 51 % of mortality for controls against 22% only for pretreated cells.
This protective effect of highly diluted cadmium occurred irrespective of the toxic dose of cadmium used. However, the pretreatment appeared less effective and statistically not significant when associated with the highest toxic concentration (5.10-5 M). The statistical analysis was performed by the ANOVA global analysis and t Student test. Similar results have been found at five different periods in the year, showing a good reproducibility of the described protective effect.
To conclude, the protective effect of very high dilutions of a metal against toxic doses of the same metal seems clearly demonstrated in proximal tubular cell cultures. Nevertheless, the pretreatment must be applied for a 120 h period. Indeed, we established that no protective effect could be observed for a 48 h pretreatment ( data not shown).
In the same way, a protective effect has been obtained with very low concentrations of cisplatin applied during 120 h against intoxication with ponderal doses of cisplatin. Whatever the cisplatin dose used for intoxication, the death rate was higher in the control than in the pretreated cell group. Hence, for the 4.10-5 M toxic dose, the percentage of dead cells decreased from 60 % for untreated cells down to 40 % for pretreated cells. Nevertheless, the index of protection was lower for cisplatin than for cadmium.
Therefore, the protective effectiveness seems to depend on the toxic dose used, the pretreatment duration and, as we can see in the figure 4, also on the height of the pretreatment dilution. For a 120 h period, we had incubated the cells with serial centesimal dilution of cadmium from 10-10 to 10-40 M. Then, cells were intoxicated with our usual ponderal range of cadmium.
For those four dilutions (10-10, 10-20, 10-30, 10-40 M), a protective index was determined. It is defined by the ratio of control cell death rate to pretreated cell one. If this index is equal to 1, there is lack of protective effect; if it is higher than 1, then it expresses a favourable effect and consequently a protective one. On the contrary, an index below 1 means an increase of cell death induced by the pretreatment itself.
As outlined in the figure 4, the protective effect of the highest dilutions is neither efficient for high toxic doses of cadmium nor for the lower ones. The index of protection is dilution dependent and it increases with the dilution from 10-10 to 10-40 M with a maximum for 10-40 M of cadmium.
The same data was obtained with another metal like cisplatin. The protection index was also dilution height dependent. However, the effectiveness of this protection was less important for the cisplatin than for cadmium. So, the most important index for cisplatin was 1.68 (2.10-5 M cisplatin) against 4.70 for cadmium ( 2 x 10-5 M cadmium).
The protective effect of highly diluted metal towards metal cytotoxicity revealed to be specific of the metal used for the dilution. We compared the effect of pretreatment with 10-40 M of cisplatin towards an intoxication with cisplatin (cisplatin versus cisplatin) and pretreatment with cisplatin towards an intoxication with cadmium (cisplatin versus cadmium). The data are shown in figure 5.
Figure
5. Protection index of cisplatin and cadmium high dilution (10-40M) in intoxicated tubular cultured cells by the same metals.
The protection index was less than 1 for each cadmium versus cisplatin experiment, which means total lack of protection. We even noted a significant decrease in death rate for the pretreated cells as compared with their control counterparts (CP vs CP): 5.10-5 M cisplatin killed 63.1 % of cisplatin pretreated cells when it killed 68.4 % of cadmium pretreated cells.
The effectiveness of the pretreatment requires the use of the same metal for high dilution and intoxication; indeed the protection induced by the pretreatment with cadmium or cisplatin dilution is significant only when the intoxication is caused by cadmium or cisplatin respectively; the principle of similarity, the basis of Homeopathy, is confirmed.
After bringing to the four experimental proofs of the effectiveness of the pretreatment by highly diluted metals in reducing cell death associated with heavy metal intoxication, we decided to study the intracellular structures, especially the organisation of cytoskeleton proteins, marker of metabolic and functional state of the cell.
By an indirect immunofluorescence technique , it is possible to visualize actin and vimentin networks respectively diffused and clearcut in the cytoplasm.
We compared the aspect and the organization of the network of both proteins in pretreated and control cells towards an intoxication by ponderal high cadmium doses (0,125, 0,25, 0,5, 2 x 10-5 M). Whatever the network studied, it seemed that the pretreatment by high dilutions is able to decrease cadmium-induced cytoskeleton protein desorganization. Indeed, for the same intoxication concentration, the network aspect of actin and vimetin appeared more normal in pretreated cells than in controls. The vimentin network seemed yet clearcut, well-organized and developed even in 24 h intoxicated cells by 0,25 x 10-5 M of cadmium (figure 6).
Figure
6 . Vimentin cytoskeleton protein organization in tubular cultured
cells (a: control non intoxicated cells, b: intoxicated non pre-treated cells,
c: intoxicated pre-treated cells.)
3. Mechanistic Approach of Heavy Metal High Dilution Protection
3.1.INFLUENCE OF VERY HIGH DILUTIONS OF CADMIUM ON INTRACELLULAR CADMIUM PENETRATION
Flame spectrophotometry allows a precise determination of cadmium concentration. The penetration rate of cadmium in the cell can be deduced from the difference between the cadmium present in the cells and the total cadmium present in the mixture and in the supernatant.
At the end of the pretreatment period (120 h), the cells were intoxicated by introducing ponderal concentrations of cadmium from cadmium standard solutions directly in the cell medium.
Figure
7. Cd penetration ratio in control intoxicated and intoxicated
pre-treated tubular cultured cells.
As we can see in figure 7, cadmium penetration in the pretreated cell group was 35 % lower than in the control group; for the lowest intoxication concentration, at 3.75 mg Cd, the penetration rate decreased from 0.36 to 0.22. For this concentration, the very high dilution seemed to reduce cadmium penetration and therefore lowered intracellular cadmium concentration which induced less dramatic cell damages.
Nonetheless, except from this concentration, no significant difference between the pretreated and control groups could be found. Furtheremore, these results are confirmed by atomic absoption , a more sensitive method for cadmium dosage.
3.2. INFLUENCE OF VERY HIGH DILUTIONS OF CADMIUM ON METALLOTHIONEIN SYNTHESIS
As previously reported by different authors, cadmium is a well-known metallothionein (MT) synthesis stimulating agent. Therefore, we wondered whether MT could be involved in the observed protective effect of highly diluted metal in heavy metal intoxication. MT was detected and quantified by radioimmunoassay (RIA) techniques by Drs LAMBOEUF and CARRERA from the laboratory of " Signalisation et Differenciation des Macrophages" ( Unité I..N.S.E.R.M. du C.H.U de Rangueil à Toulouse).
Figure
8 . Metallothionein synthesis in intoxicated control and intoxicated
pretreated tubular cultured cells.
Figure 8 shows clearly that cadmium induces metallothionein synthesis in renal tubular cell culture, and that the pretreatment with very high dilutions of cadmium enhances metallothionein biosynthesis after a 24 h cadmium intoxication. The 120 h pretreatment with 10-30 M dilution increases significantly the concentration of metallothionein as compared with the control group.
After 24 hour incubation with 0.25 x 10-5 M of cadmium, the concentration of MT decreases from 1.93 mg MT / mg of protein for control cells to 2.67 mg MT / mg protein for pretreated cells. In the same way, 0.5 x 10-5 M cadmium induces the synthesis of 4.0 mg of MT / mg protein in the control cell group against 6.03 mg of MT/ mg protein in the pretreated cell group.
So, it seems that very high dilutions of cadmium enhance metallothionein biosynthesis consecutively to a ponderal intoxication with cadmium; this MT biosynthesis seems to remain dilution-dependent. Indeed, data not shown here, revealed that a solution of 10-10 M cadmium still induces metallothionein sythesis but less than cadmium 10-20 and 10-30 M solutions. The pretreatment with 10-20 M favours MT synthesis but not enough for protecting against an intoxication with 0.5 x 10-5 M cadmium.
In fact, it seems that only the pretreatment with cadmium 10-30 M dilution has a protective effect, which could explain its ability to enhance MT biosynthesis consecutively to ponderal intoxication of cadmium.
The presentation of the results obtained in our laboratory for more than 12 years has been divided into two chronological and methodological parts, considering previous in vivo and recent in vitro experiments.
We have shown, in vivo in rodents, that high dilutions of mercury can
significantly reduce the death rate induced by toxic doses of the same metal.
Thus, the death rate of mice intoxicated with high doses of mercury was
markedly reduced by a 7 day pretreatment with 10-30 M concentrations of that metal. For example, the death
rate following a single injection of 5 mg/kg HgCl2 was 73.4% in control, 50% for those
pretreated with 10-18
M and only 26.7% for those pretreated with 10-30 M.
We found the same
profile of response with our in vitro
model in which 10-30
and 10-40 M dilutions provided a fair protection
against cadmium induced cytotoxicity in renal tubular cell cultures. Similar
results have recently been described for high dilutions of thymulin (Bastide et al., 1987), bursin (Youbicier-Simo et al.., 1993) and silica (Oberbaum et al.., 1992).
The classical neutral
red test permits a standardization of cytotoxicity evaluation procedures using
renal cell cultures intoxicated by toxic cadmium solutions. Our results are reproducible (5/5 for 10-30 M and 4/4 for 10-40
M). This in vitro model
using cell cultures eliminates numerous sources of variability, especially
chronobiological ones, present in in vivo
tests. Our results are confirmed by similar in
vitro experiments in other cultured mammalian cells intoxicated by cadmium
and protected by low dilutions of the same metal (Van Wijk and Wiegant , 1994).
The protective effect of
very high cadmium dilutions against toxic doses of this metal seems clearly
demonstrated. Similar protection against cisplatin intoxication is also
demonstrated for ultra low doses of cisplatin. This protection seems specific
since cadmium dilutions do not protect against cisplatin toxicity despite the
fact that cisplatin is also a tubulotoxic metal.
Recent experiments
carried out in our laboratory have shown that MT synthesis was increased in Cd
intoxicated cells after exposure to very high Cd dilutions (10-20, or 10-30 and 10-40 M) for 120 hours.
Similar results have been reported by Pellegrini et al. (1994) with low doses of cadmium (1 mM to 0.1 nM) in an immature human T cell
line. A 24 h pretreatment with non toxic concentrations of cadmium confered
protection against subsequent cytotoxic doses and was able to induce
metallothionein - IIa gene expression. An increase of MT synthesis has been
already observed with toxic or subtoxic concentrations of heavy metals such as
zinc, cisplatin or cadmium. These results are in fair accordance with other
ones from our laboratory showing that such a treatment decreases the metal
storage in the cell by, for example, changes in membrane permeability.
These in vitro data support our in vivo results suggesting that very
high dilutions of metals may be useful in decreasing or even eliminating the
toxic effects of nephrotic metals in patients chronically exposed to such
products. Such a protective procedure was already well accepted for pretreatments
with milligram or microgram doses in
experimental toxicology (Flora et
al., 1982a,1982b; Stacey and Klaassen, 1981; Magos et al., 1979; Yamane and Koizumi, 1982).
These data demonstrate
not only the relevance of in vitro models
in this area of toxicology but also confirm the specific protective effects of
high dilutions of cadmium or cisplatin against subsequent cytotoxic doses of
the same metals.
Aknowmedgements
We thank
Dr P. Dorfman for assistance with statistical analysis and helpful and kind
discussion. These studies were supported by a grant of Laboratoires Dolisos
with a CIFRE contract with the French Ministery of Research and Technology.
5. References
Bastide, M., Daurat, V.,
Doucet-Jaboeuf, M., Pelegrin, A. and Dorfman P. (1987) Immunomodulatory
activity of very low doses of thymulin in mice, Int. J. Immmunotherapy 3, 191-200.
Bruggeman, I.M., Mertens, J.J.,
Temmink, J.H., Lans, M.C., Vosr,.M. and
Van Bladeren, P.J. (1989) Use of monolayers of primary rat kidney cortex cells
for nephrotoxic studies, Toxicol. Vitro 3,
261-269.
Cal, J.C., Larue, F.,
Guillemain, J. and Cambar, J. (1986) Chronobiological approach of protective
effect of Mercurius corrosivus
against mercury induced nephrotoxicity, Ann.
Rev. Chronopharmacol. 3, 99-103.
Cal, J.C., Larue, F.,
Guillemain, J. and Cambar, J (1988) Chronobiological approach of mercury
induced toxicity and the protective effect of high dilutions of mercury against
mercury-induced nephrotoxicity, in A. Guillouzo
(ed.) Liver Cells and Drugs, John
Libbey Eurotext Publisher, Paris, pp 481-485.
Cambar, J., Delbancut, A. and
Dorfman, P (1994) Applications des très hautes dilutions en toxicologie, Cahiers de Biothérapie, 125,
27-34.
Cazin, J.C., Cazin, M.,
Gaborit, J.L., Chaoui, A., Boiron, J., Belon, P., Cherruault, Y., and
Papayanotou, C.(1987) A study of the effect of decimal and centesimal dilutions
of arsenic on the retention and mobilization of arsenic in the rat, Human Toxicol. 6, 315-320.
Fisher, P., House, I., Belon,
P. and Turner, P. (1987) The influence of the homeopathic remedy Plumbum met
on the excretion kinetics of lead in rats, Human Toxicol. 6,
321-324.
Flora, S.J.S., Behari, J.R.,
Ashquin, M. and Tandon, S.K. (1982a) Time dependent protective effect of
selenium against cadmium-induced nephrotoxicity and hepatoxicity, Chem. Biol. Inter., 42, 345-351.
Flora, S.J.S ., Jain, V.K.,
Berahi, J.R. and Tandon, S.K. (1982b) Protective effect of trace metals in lead
intoxication, Toxicol. Letters 13, 51-56.
Herkovits, J. and Perez-Coll,
C. (1991) Potentized microdoses of cadmium reduce the lethal effect of this
heavy metal in amphibian embryos, Berlin
J. Res. Homeo. 1, 93-96.
Humes, H.D. and Weinberg, J. M.
(1986) Toxic nephropathy, in B.M. Brenner and F.C. Rector (eds) The Kidney, Saunders Publisher,
Philadelphia, pp 1491-2008.
Ito, T. and Sawauchi, K. (1966)
Inhibitory effects on cadmium-induced testicular damage by pretreatment with
smaller cadmium dose, Okajima. Fol. Anat.
Jap., 42, 107-117.
Lagroye, I., L'Azou, B.,
Lakhdar, B., Plande, J. and Cambar, J. (1995) Nephrotoxicité in vitro: modèles
glomérulaires, in M. Adolphe and A. Guillouzo (eds) Toxicologie cellulaire in vitro , Editions INSERM Publisher, Paris,
pp 121-150.
Larue, F., Cal, J.C., Dorian,
C., Guillemain, J. and Cambar, J. (1987) Influence du prétraitement de
dilutions infinitésimales de Mercurius
corrosivus sur la mortalité induite par le chlorure mercurique, Néphrologie, 6, 86.
Linde, K., Jonas, W.B.,
Melchart, D., Worku, F., Wagner, H. and
Eitel F. (1994). Critical review and meta-analysis of serial agitated
dilutions in experimental toxicology, Human Exp.Tox. 5, pp 481-492.
Magos, L., Webb, M. and
Buttler, M. (1979) The effect of Cd pretreatment on the nephrotoxic action and
kidney uptake of mercury in male and female rats, Brit J Pathol, 55,
589-594.
Mery, J.P and Fillastre, J.P.
(1983) Reins et médicaments, in J. Hamburger, J.P. Grunfeld (eds.), Nephrologie, Flammarion, Paris, pp
1193-1207.
Oberbaum, M., Markovits, R.,
Weissman, Z., Klinkevits, A. and Bentwich, Z. (1992) Wound healing by
homeopathic silica dilutions in mice,
Harefuah, 123, 79-82.
Pellegrini, O., Davenas, E., El
Azzouzi, B., Jurgens, P., Benneniste, J., Manuel, Y. and Thomas, Y. (1994)
Stress proteins in human lymphocytes. Induction of metallothionein and heat
shock protein genes by cadmium in an immature T cell line, Cellular Pharmacology, 1,
91-97.
Stacey, M.A. and Klaasen, C.D.
(1981) Interaction of metal ions with cadmium-induced cellular toxicity, J. Toxicol. Environ. Health 7, 149.
Terhar, C.J., Vis, E.,
Roudabush, R.L. and Fasset, D.W. (1965) Protective effects of low doses of
cadmium chloride against subsequent high oral doses in the rat, Toxicol. Appl .Pharm. 7, 500.
Toutain, H.J.- Néphrotoxicité
in vitro: modèles de cultures de cellules tubulaires rénales (1995). in M. Adolphe and A. Guillouzo (eds) Toxicologie cellulaire in vitro ,
Editions INSERM Publisher, Paris, pp 151-189.
Van Wijk, R. and Wiegant,
F.A.C. (1994) Cultured mammalian cells in
homeopathy research; The similia principle in self-recovery. Utrecht
University Publisher, Utrecht.
Yamane, Y. and Koizumi, T.
(1982) Protective effect of Mo on acute toxicology of HgCl2, Toxicol. Pharmacol. 15, 14-221.
Yoshikawa, H. (1973) Preventive
effect of pretreatment with cadmium on acute cadmium poisoning in rats Industrial health (Japan) 11, 13-119.
Youbicier-Simo, B.J., Boudard,
F., Mekaouche, M., Bastide, M. and Baylé, J.D. (1993) Effects of embryonic
bursectomy and in ovo administration of highly diluted bursin on
adrenocorticotropic and immune responses of chickens, Int. J. Imunotherapy 9, 169-180.