Effects of acute administration of ayahuasca on the oxidative stress and neuronal apoptosis in rats

Introduction : The y hu sc h ve leg l uthoriz tion for religious use nd scientific rese rch. There re few published studies to determine their neurotoxic risk.

Objective : Ev lu te the potenti l toxicity of y hu sc in Wist r r ts on the neuron l poptosis nd its rel tion to systemic effects fl gs of oxid tive stress.

Method : Neurotoxicity w s ssessed by y hu sc occurrence of neuron l poptosis by fluorescence of C sp se-3 nd by TUNEL ss y. During 21 d ys, by g v ge, twelve Wist r r ts received 2 ml of 50% y hu sc nd the control group received 2 ml of w ter. Glut thione, m lon ldehyde nd vit min E n lyzes were performed to ev lu te serum nd hep tic lipid peroxid tion s well s cre tinine nd ure n lyzes for ssessing kidney function.

Results : The c sp se-3 showed no differences between the two groups. The v lues of serum MDA, serum glut thione nd hep tic vit min E showed st tistic lly signific nt reduction in the group tre ted with y hu sc . R ts tre ted with y hu sc lso h d higher me n v lue st tistic lly signific nt poptotic neurons, me sured by TUNEL ss y.

Conclusions: The results of this rese rch indic te the presence of process of oxid tive stress in r ts tre ted with y hu sc , with st tistic lly signific nt findings neuron l poptosis ss y with TUNEL. The reduced levels of serum MDA in y hu sc group could point to prob ble neuroprotective effect, however were lso ccomp nied by reduction in GSH nd vit min E, which indic tes the occurrence of incre sed oxid tive stress in this group.

Despite the tr dition of its use in sh m nic contexts, the use of y hu sc ( y hu sc ) h s c used much controversy. In 1987, the subst nce w s leg lized offici lly, nd in 2006, in Br zil CONAD uthorized its religious use nd for scientific rese rch (CONAD, 2006).

There re few published studies th t ev lu te nd determine the neurotoxic risk y hu sc in experiment l nim ls, specific lly in poptotic processes, nd there is still much controversy between the neuroprotective nd neurotoxic effects of its components.

Ay hu sc is h llucinogenic drink, whose m in components re h rmine-HRM, h rm line-HRL, the tetr hydro-h rmine-HHT nd N, N-dimethyltrypt mine-DMT (McKenn et al. , 1984 ; McKenn et al. , 1984b; Freedl nd, M nsb ch, 1999; Oliveir et al. , 2010).

DMT is serotonin psychotropic gent th t c n c use ch nges in perception of re lity with ment l complex form tion im ges (McKenn et al. , 1984 ; McKenn et al. , 1984b; C ll w y et al. , 1999; C rlini, 2003). Other neurophysiologic l effects of the bet -c rbolines include competitive inhibition of dop mine, epinephrine, nd norepinephrine in syn ptosomes; in the synthesis of biogenic mines nd possible role in benzodi zepines receptors (McKenn , 2004).

Sever l uthors h ve described the neuroprotective properties of β-c rbolines (Lee et al. , 2000; Kim et al. , 2001; P rk et al. , 2003; Mour et al. , 2007) but lso s neurotoxins h ve been proposed, p rticul rly for d m ge to mitochondri (Uezono et al. , 2001; Chen et l, 2005; Song et al. , 2006).

The dimetiltript mine h s been considered neurotoxic nd since 1971 is included in Level I of the Convention of the United N tions Psychotropic Subst nces (Pires et l,. 2010).

The im of this study w s to cre te n explor tory process th t would llow ev lu te the toxic potenti l of y hu sc in Wist r r ts s the p r meters of oxid tive stress, s well s its rel tionship to neuron l poptosis.

MATERIALS AND METHODS

Obtaining composition and dosage of ayahuasca

The y hu sc used in this work w s obt ined from the L bor tory of Toxicologic l (LAT), F culty of

Ph rm ceutic l Sciences USP in 2008. A te m of rese rchers in the l bor tory extr cted lk loids by using solid ph se nd g s chrom togr phy with detector nitrogen- phosphorus (GC-NPD) reported the determin tion of its constituents, n mely: DMT = 0.42mg / ml; HRM = 1.37 mg / ml; HRL = 0.62 mg / ml; HHT = 0.35 mg / 16 ml.

One study ex mined the effects of y hu sc in r ts concluded th t dministr tion of dose of 500 mg / kg did not produce neurologic l imp irment. The uthors defined the LD 50 in r ts of y hu sc t 5.83 g / kg. This s me dos ge nd equiv lent p r meters determined were used in this study the dministr tion of the y hu sc experiment l group (Souz -Brito et al. , 2010) (17).

Animals and experimental conditions

Twenty four Wist r m le r ts with initi l weight of pproxim tely 200 g, provided by the Centr l Anim l Biotery of USP-RP were r ndomly divided into two groups of twelve nim ls e ch. They were pl ced in groups of four in pl stic boxes with six met llic grid with food nd w ter ad libitum . They were housed in the nim l house of the Dep rtment of Intern l Medicine of FMRP/USP, with cycle of 12 hours light-d rk, light off t 18:00 hours, nd controlled const nt temper ture of 24-26ºC, p ssing through n djustment period of two d ys .

Twelve r ts were given by g v ge d ily or l dose of 2 ml of 50% y hu sc ( y hu sc group), while the control group received 2 ml of drinking w ter for twenty-one d ys. It w s then weekly routine weighing nd urine collection in met bolic c ges for 24 hours.

At the end of tre tment, nim ls were euth nized by dec pit tion. They were extr cted the br in, liver nd whole blood from e ch nim l for biochemic l n lysis in L bor tory of Nutrition nd Met bolism of the Dep rtment of Intern l Medicine of Ribeirão Preto-USP.

Coron l sections were performed on hippoc mp l region ( pproxim tely bregm -3.80 mm), nd the obt ined section w s pl ced in 4% p r form ldehyde. inclusion histologic l procedures were performed on p r ffin; Microtome t 5 um nd st ined with hem toxylin-eosin being ssembled the bl de with gl ss coverslips with sections through Entell n (Merck ®). The rem ining br in tissue w s pl ced in the middle of homogeniz tion to br in, ground nd frozen t -18 ° C. The whole blood w s removed nd centrifuged t 3500 rpm for 10 minutes nd the serum frozen for subsequent n lysis obt ined Glut thione (GSH), m lon ldehyde (MDA) nd vit min E.

Losses Sampling

The s mple size of the control group on Frid y weighing decre sed to ten subjects, due to the de th of two mice in this group. At necropsy performed there h s been no signific nt finding. The s me situ tion h ppened to mouse y hu sc group on the d y of s crifice, which is why the number of this group ppe rs s twelve in some n lyzes nd eleven others. In the ev lu tion of the tunnel, the s mple number of both groups decre sed to six due to the impossibility of rem ining br in s mples for neuron l poptosis n lysis.

Biochemical Methods

Quantification of total protein

By the Biuret method nd using colorimetric commerci l kit (L btest®).

Qquantification of reduced glutathione-GSH

GSH w s qu ntified by colorimetric method, which consists in the re ction of this molecule with 5,5'-dithiobis-2-nitrobenzoic cid (DTNB), nd subsequent re ding in spectrophotometer.

For qu ntific tion of serum GSH w s dded 1 mL of Tris-EDTA 25 ul of e ch serum s mple in test tube p cked on ice; homogenized s mple w s performed nd bsorb nce re d t w velength of 412 nm, th t subsequently were discounted from the second bsorb nce re ding, in order to elimin te serum interference. Then the s mples were poured into respective test tubes nd 25 ul were dded DTNB. After sh ke them, the bsorb nce re d t 412 nm. The s me procedure w s performed with bl nk which did not h ve the serum s mple. The concentr tion w s c lcul ted using st nd rd curve of GSH-EDTA (0.02M) with known concentr tions of GSH e ch re ding.

Quantification of Malondialdehyde

W s performed by determin tion of thiob rbituric cid re ctive subst nces s follows: dded one ml of 1.15% KCl 100 mg tissue; m cer ted nd the mixture were dded 2 ml solution of TBA-TCA-HCl (15% trichloro cetic cid, 0.375% thiob rbituric cid nd 0.25 N hydrochloric cid); The mixture w s he ted for fifteen minutes in boiling w ter b th, cooled nd centrifuged for ten minutes t 3000 rpm t room temper ture nd the supern t nt used for re ding t 535 nm in spectrophotometer.

To 100 ul of serum w s dded 200 of TBA-TCA-HCl. This mixture w s he ted for fifteen minutes in boiling w ter b th, cooled nd centrifuged t 1000 g for ten minutes t room temper ture. W s obt ined supern t nt w s used for determin tion of bsorb nce t 535 nm. White used consisted solely by solution TBA-TCA-HCl.

For the definition of MDA concentr tion w s pplied mol r bsorption coefficient of the product (E535 = 1,56x105M-1 cm-1). A st nd rd curve w s performed using stock solutions of MDA. The concentr tions found in the s mples were within this curve shows good line rity with the st nd rd.

Quantification of Vitamin E

It w s performed in liver nd serum s mples by high-perform nce liquid chrom togr phy (HPLC) (Shim dzu chrom togr ph equipped with detector UV / Visible). The equipment w s c libr ted with α-tocopherol st nd rd solutions (Sigm ®), nd before e ch re ding w s injected into st nd rd α-tocopherol solution. The concentr tion of vit min E in the s mples w s c lcul ted using the r tio of the re of the n lyte nd the intern l st nd rd, fter obt ining c libr tion curve with known concentr tions.

Quantitation of urinary creatinine

The kit Doles® modified J ffe colorimetric system w s used for cre tinine qu ntific tion b sed on the principle th t this re cts with picr te in lk line medium, forming reddish-yellow colored complex.

Quantitation of urinary urea

For the determin tion of ure in urine of the r ts used the EC Ure L betest enzym tic-colorimetric system. The principle of this method is to hydrolyze the ure by the ure se to mmonium ions nd CO2. The mmonium ions re ct in lk line pH with s licyl te nd sodium hypochlorite under the c t lytic ction of sodium nitroprusside to form indophenol blue.

Method laboratory for apoptosis neuronal Caspase-3 Assay

The results were expressed s fluorescence v lues for the kit does not supply the c sp se-3 st nd rd.

TUNEL assay

A front l section of e ch br in tissue w s fixed immedi tely in 4% p r form ldehyde for five d ys. one second cut w s more loc lized in the hippoc mp l region. For e ch section, two slides were prep red. For the detection of neuron l cells with DNA d m ge, induced by the use of y hu sc , we used the TUNEL ss y (Kit Colorimetric TUNEL System (Promeg ®) detecting the fr gment tion of DNA (G vrieli et al. , 1992).

The fr gmented DNA c n be histochemic lly n lyzed with light microscopy by st ining brown present in the cytopl sm nd nucleus of cells. Only the neuron l cell bodies were n lyzed nd the results expressed in number of poptotic cells visu lized by re . Ten microscopic fields t 40x m gnific tion were observed nd used for counting poptotic neuron l bodies. For this n lysis we used the AxioVision® softw re (version 4.7.2). To obt in the im ges of the fields n lyzed we used Zeiss m chine with c pture c mer JVC TK-1270.

Statiscal Analysis

Descriptive st tistics (me n, minimum nd m ximum st nd rd devi tion) for ch r cterizing the s mple ccording to group ( y hu sc nd control) nd v ri bles body weight ch nges, org n weight, ren l function tests, nd lipid peroxid tion neurotoxicity were ev lu ted.

For n lysis of dependent v ri bles (tre tment with y hu sc ) nd independent (neurotoxicity) we used the Chi-squ re test to ev lu te the difference in proportions between both groups, s well s p r meters indic tive of oxid tive stress. For ev lu ting the me n difference between y hu sc nd control groups we used the non-p r metric M nn-Whitney test. For the n lysis of correl tion between v ri bles w s used the Spe rm n correl tion test. The signific nce level w s 5% for ll tests (p ≤ 0.05).

For st tistic l n lysis we used the St tistic l P ck ge for Soci l Science (SPSS) version 15.0.

RESULTS

The group tre ted with y hu sc showed lower v lues th n the control group, expressed s me n of weight g in nd percent ge g in; The st nd rd devi tion of both v ri bles w s higher in y hu sc group, but no differences were st tistic lly signific nt (T ble 1).

The three me surements of urin ry ure in mg / dl were higher in y hu sc group comp red to the control group, nd the v lue of the third me surement st tistic lly signific nt (T ble 2).

Hep tic MDA h d higher v lues in control r ts; They were observed higher GSH v lues in y hu sc group nd vit min E is m rkedly diminished in nim ls of this group showing st tistic lly signific nt (T ble 3).

Serum levels of MDA, GSH nd vit min E re decre sed in the group receiving the y hu sc , with st tistic l signific nce for MDA nd GSH. The serum vit min E v lue were not st tistic lly signific nt (T ble 4).

The C sp se-3 v lues w s lower in y hu sc group, but no st tistic l signific nce. The TUNEL ss y v lues re lso lower in the control group   nd y hu sc h ve st tistic lly signific nt v lues for neuron l poptosis (T ble 5).

Table 1. Weightg in v lues nd g in percent ge of y hu sc nd control groups, expressed in gr ms.

	Ayahuasca group (n = 12)			Control Group (n = 10)
	M	DP	Min. - Max.	M	DP	Min. - Max.	U	P
Weight gain	112.83	28.45	67.00 to 169.00	118.60	15.15	99.00 to 148.00	52.00	0.60
gain percentage	55.82	13,90	35.62 to 82.84	57.91	7.94	46.70 to 74.75	54.00	0.69

V lues expressed s me n (m); St nd rd Devi tion (SD) nd minimum nd m ximum v lues min-m x.); Coefficient U = M nn-Whitney; p = st tistic l signific nce.

Table 2 .Ure levels in urine s mples(n=3) of y hu sc nd control groups, expressed in mg / dL

Weekly measurements of urinary urea	Ayahuasca group				Group control
	n	M	DP	Min. - Max.	n	M	DP	Min. - Max.	P
1	12	93.33	32.38	35.00 to 140.00	12	74.01	29.35	30.62 to 131.25	0.18
2	12	82.14	22,40	35.00 to 131.25	12	69.27	48.14	26.25 to 201.25	0.09
3	12	88.59	32.75	35.00 to 131.25	10	43.75	26.41	8.75 to 96.25	<0.01

Amounts of s mpling (n); Medium (M); St nd rd Devi tion (SD) nd minimum nd m ximum v lues (min-m x.); p = st tistic l signific nce.

Table 3 . Hep tic levels of MDA (nmol / g protein), GSH (mol / g protein) nd vit min E (nmol /g tissue) in control or Ay hu sc groups.

Ayahuasca group (n = 11)						Control Group (n = 10)
	M	md	DP	Min. - Max.	M	md	DP	Min. - Max.	U	P
MDA	37.84	34.47	7.40	30.13 to 52.60	43.36	45.48	6.75	31.20 to 50.37	32,00	0.10
GSH	5.60	5.66	0.64	4.84 to 6.80	5.34	5.51	0.67	4.00 to 6.13	45.00	0.48
Vit. E	41.91	41.31	10.16	18.70 to 55.36	80.88	78.34	21.71	48.96 to 118.58	4.00	<0.01

V lues expressed s me n (m); Medi n (Md); St nd rd Devi tion (SD) nd minimum nd m ximum v lues (Min -M x..); Coefficient U = M nn-Whitney; p = st tistic l signific nce.

Table 4. Serum levels of MDA (nmol / g protein), GSH (mmol / g protein) nd vit min E (nmol / ml) in control or Ay hu sc groups.

Ayahuasca group (n = 11)             Control Group (n = 10)

	M	md	DP	Min. - Max.	M	md	DP	Min. - Max.	U	P
MDA	26.60	25.35	4.50	21.12 to 33.08	30.70	29.95	4.60	25.60 to 38.46	27.00	0.05
GSH	0.61	0.71	0.35	0.04 to 1.30	1.20	1.18	0.32	0.81 to 1.80	9.50	<0.01
Vit E	4.69	4.18	1.56	2.89 to 8.19	6.20	5.65	2.32	3.58 to 11.41	29,00	0.07

V lues expressed s me n (m); Medi n (Md); St nd rd Devi tion (SD) nd minimum nd m ximum v lues (Min -M x..); Coefficient U = M nn-Whitney; p = st tistic l signific nce.

Table 5 V lues of c sp se-3 nd TUNEL in control or Ay hu sc groups, expressed in number nd level of fluorescence positive cells, respectively.

Ayahuasca group                      Group control

	n	M	DP	Min. - Max.	n	M	DP	Min. - Max.	P
Caspase-3	11	25.51	6.89	16.39 to 37.94	10	27.33	6.14	17.69 to 39.64	0.40
TUNEL	6	1.33	0.52	1.00 to 2.00	6	3.00	2.00	1.00 to 6.00	0.05

Amounts of s mpling (n); Medium (M); St nd rd Devi tion (SD) nd minimum nd m ximum v lues (Min. - M x.). p = st tistic l signific nce.

DISCUSSION

There re some limit tions found in this study: the

difficulty of finding public tions of previous studies on the neurotoxic effects of y hu sc in experiment l nim ls; bsence of specific nd recognized nim l model for this type of rese rch; using n tur l compound inste d of st nd rd synthetic ctive ingredients; l ck of beh vior l studies of nim ls fter the use of y hu sc ; compound m n gement techniques, bec use there is still no ccur te knowledge of the ph rm cokinetics nd ph rm codyn mics of y hu sc in Wist r r ts.

The results obt ined in this study, there w s difference in me n weight for tre ted mice lost more weight y hu sc comp red to the control group. A toxicity study with pregn t Wist r r ts tre ted with y hu sc reports signific nt loss v lues of m tern l weight, which w s interpreted s sign of toxicity of this subst nce (Oliveir et al. , 2010).

Another study rel ted weight loss nd m lnutrition, depletion of GSH nd oxid tive stress (Wu et al. , 2004). As for the kidney function p r meters, urin ry ure me surement showed higher v lues with st tistic l signific nce in the group of r ts tre ted with y hu sc , ren´t other studies with nim l models tre ted with y hu sc th t v lid te these biochemic l p r meters.

It is considered th t the ch nges in the v lues of ure nd cre tinine urin ry m y represent sign of ren l d m ge in r ts tre ted with y hu sc . The n lysis of kidney function re fund ment lly import nt p r meters in toxicity studies in nim ls nd hum ns, since dehydr tion st tes, or stress the nim ls could, t some point, provide or initi te n poptotic process.

Reg rding the indic tors of oxid tive stress, comp r tive n lyzes of serum levels of GSH nd MDA well s hep tic vit min E showed st tistic lly signific nt decre se in mice tre ted with y hu sc .

Recent studies underscore the import nce of n en bling intr cellul r environment to the ch nges in regul tion of poptosis in the redox environment nd consider the depletion of GSH common fe ture of cell de th poptosis triggered by wide v riety of stimuli including: ctiv tion of de th receptors, stress gents environment l nd cytotoxic drugs. This depletion of GSH, considered initi lly only s byproduct of oxid tive stress gener ted during cell de th, is now seen s critic l regul tor of poptosis. Thus, st tistic lly signific nt reduction of serum levels of GSH in the group of r ts tre ted with y hu sc m y indic te th t oxid tive stress m y be contributing to direct regul tion of neuron l poptosis. The poptotic process is regul ted by c sc des intrinsic nd extrinsic sign ling. It is demonstr ted th t depletion of GSH c n ctiv te both poptotic sign ling p thw ys in sever l control points, predisposing cells to poptosis or directly triggering the de th cellul r (Fr nco, Cidlowsky, 2009). Also the MDA elev ted in v rious dise ses nd intoxic tion, it h s been linked to d m ge c used by free r dic ls, produced by lipid peroxid tion processes (M teos et al., 2005; Antunes et al., 2008).

Unlike the results reported in the liter ture in this study were found reduced serum nd hep tic MDA levels in the group of r ts tre ted with y hu sc , which could be n indic tion of proper ntioxid nt ction mech nism of the subst nce prob bly the content of ß-c rbolines bec use this molecule h ve been described s neuroprotective.

With respect to vit min E, they were found signific ntly decre sed levels nd st tistic l signific nce n lysis in liver of r ts tre ted with y hu sc . Vit min E is n import nt ntioxid nt th t is consumed in n ttempt to void cellul r imb l nce oxid tive stress (Jordão et al. , 1998) nd decre sed v lues could suggest the presence of oxid tive stress induced by y hu sc consumption. At the moment there re no other published studies on the occurrence of this phenomenon (Jordão et al. , 1998).

The TUNEL ss y c rried out for ev lu tion of the occurrence of neuron l poptosis, the group of r ts tre ted with y hu sc h d higher me n v lue st tistic lly signific nt poptotic neurons. In study rel ting poptosis nd oxid tive stress, or uthors suggest th t oxid tive stress m y interfere with the ctiv tion of c sp ses nd throughout the poptotic process by depletion of denosine triphosph te (ATP) intr cellul r nd m y even derive it to necrosis (Lee, Sh cter, 1999).

B sed on the results of biochemic l n lyzes of mice tre ted with y hu sc in the present study, we consider the possibility of n oxid tive stress process, nd perh ps for this re son neuron l poptosis is incre sed in this group of nim ls.

Another w y to neurotoxicity produced by use of y hu sc could be the existence of cell microinjuries c used by cerebr l ischemi / reperfusion. These lesions could initi te complex series of biochemic l nd molecul r events with loss of function by disruption of the cell integrity due to glut m tergic excitotoxicity, the ionic imb l nce, the re ctions of free r dic ls nd poly polymer se ctiv tion- PARP (Zh ng et al., 2009). This is the protein th t binds to DNA protects its integrity nd f cilit ting the rep ir of ruptures in their ch ins nd is involved in import nt biologic l processes such s: genome integrity monitoring, response initi tion DNA d m ge nd poptosis (Is belle et al., 2010).

However others uthors showed the ther peutic properties of y hu sc components in ischemi /reperfusion injury of the eye (Szilágyi et al. , 2022).

Ayuh sc tre tment counter cted biochemic l lter tions, s MDA nd nitrite levels in model of depression elicited by unpredict ble chronic mild stress but did not displ y ny lter tions in non-stressed r ts (X vier et al. , 2021).

In one recent study y hu sc showed s promising ppro ch to prevent sepsis-induced neuroinfl mm tory nd oxid tive stress, with reduced v lues of thiob rbituric cid re ctive subst nces (de C m rgo et al. , 2025).

The y hu sc int ke m y h ve interfered with the poptotic process in r ts for sever l re sons: ch nge in cell structure due to ischemi ; cytotoxicity of oxid tive stress second ry to depletion of GSH nd vit min E; glut m tergic or possible biochemic l mech nisms.

B sed on the results obt ined, it c n be s id th t there is neurotoxic rel ted to the use of y hu sc process in the group of r ts submitted to sub cute tre tment with this subst nce. The lower v lues of serum GSH nd vit min E hep tic found in this group of nim ls, s well s decre sed body weight nd incre sed urin ry ure , comp red to the control group v lues c n be evidence of systemic sign ling effects of stress oxid tive nd s result, is indirectly imp cting the ctu tion or the stimul tion of br in poptotic process.

CONCLUSION

The unique findings of the present study th t could hypothetic lly be indic tive of neuroprotection induced by the use of y hu sc re the v lues decre sed MDA serum nd liver, but not st tistic lly signific nt, nd t this time it is not possible to c tegoric lly st te th t the y hu sc h s neuroprotective properties bec use s lre dy highlighted with lower v lues were lso ccomp nied by reduction in GSH ntioxid nts nd vit min E, which indic tes the occurrence of incre sed oxid tive stress in this group or t le st gre ter depletion of ntioxid nts, is necess ry future studies in order to incre se knowledge rel ted to possible ther peutic or deleterious effects of y hu sc .

CONFLICTS OF INTEREST

The uthors decl re th t they h ve no potenti l conflicts of interest.