Deletion of glutathione-s-transferase m1 reduces azathioprine metabolite concentrations in young patients with inflammatory bowel disease.

GOALS: To investigate, in young patients with inflammatory bowel disease (IBD) treated with azathioprine, the association between genetic polymorphisms of thiopurine-S-methyl-transferase (TPMT), inosine-triphosphate-pyrophosphatase (ITPA), and glutathione-S-transferases (GST), involved in azathioprine metabolism, the concentration of the main metabolites of azathioprine, thioguanine nucleotides (TGNs) and the methylated nucleotides (MMPN), and the dose of the medication. BACKGROUND: Azathioprine is widely used in IBD as an immunosuppressive agent, particularly to maintain remission in patients with steroid refractory disease. Azathioprine is a prodrug and requires conversion to its active form mercaptopurine, which has no intrinsic activity, and is activated by the enzymes of the purine salvage pathway to TGNs. Polymorphisms in genes of enzymes involved in azathioprine metabolism influence the efficacy and toxicity of treatment. STUDY: Seventy-five young patients with IBD treated with azathioprine at least for 3 months were enrolled and genotyped for the selected genes; for these patients, TGN and MMPN metabolites were measured by high performance liquid chromatography in erythrocytes. RESULTS: GST-M1 deletion was associated with lower TGN/dose ratio (P=0.0030), higher azathioprine dose requirement (P=0.022), and reduced response to therapy (P=0.0022). TPMT variant genotype was associated with lower MMPN concentration (P=0.0064) and increased TGN/dose ratio (P=0.0035). ITPA C94A polymorphism resulted in an increased MMPN concentration (P=0.037). CONCLUSIONS: This study describes the effect of candidate genetic polymorphisms in TPMT, ITPA, and GST-M1 on azathioprine pharmacokinetics in IBD patients, showing, for the first time, relevant effects of GST-M1 genotype on azathioprine metabolites concentration.

Association between BclI polymorphism in the NR3C1 gene and in vitro individual variations in lymphocyte responses to methylprednisolone.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: In vitro lymphocyte steroid sensitivity has been suggested as a useful tool to predict in vivo response to glucocorticoid treatment in different inflammatory chronic diseases. A correlation between genetic polymorphisms and clinical response to glucocorticoids has been demonstrated in these patients. WHAT THIS STUDY ADDS: The BclI polymorphism in the glucocorticoid receptor (NR3C1) gene is associated with higher methylprednisolone potency in vitro. The combined evaluation of the in vitro sensitivity to methylprednisolone and BclI polymorphism could represent an aid for physicians to adjust therapy a priori. AIM To evaluate the association between the in vitro sensitivity of peripheral blood mononuclear cells (PBMCs) to methylprednisolone (MP) and the presence of genetic polymorphisms involved in glucocorticoid (GC) response. METHODS: In vitro MP inhibition of the proliferation of lymphocytes stimulated with concanavalin A was determined. Non linear regression of dose-response data was performed computing the MP concentration required to reduce proliferation to 50% (IC(50) ). The maximum inhibition achievable at the highest MP concentration (I(max) ) was also calculated. Moreover, the Taqman technique was used to analyze the BclI polymorphism in the NR3C1 gene and the Leu155His polymorphism in the NALP1 gene. RESULTS: A significant association between the BclI mutated genotype and an increased in vitro sensitivity to GCs was observed. CONCLUSIONS: The a priori evaluation of the BclI polymorphism, associated with a lymphocyte proliferation assay, could represent a useful diagnostic tool for the optimization of steroid treatment.

Genetic predictors of glucocorticoid response in pediatric patients with inflammatory bowel diseases.

BACKGROUND: Glucocorticoids (GCs) are used in moderate-to-severe inflammatory bowel diseases (IBD) but their effect is often unpredictable. AIM: To determine the influence of 4 polymorphisms in the GC receptor [nuclear receptor subfamily 3, group C, member 1 (NR3C1)], interleukin-1ß (IL-1ß), and NACHT leucine-rich-repeat protein 1 (NALP1) genes, on the clinical response to steroids in pediatric patients with IBD. METHODS: One hundred fifty-four young IBD patients treated with GCs for at least 30 days and with a minimum follow-up of 1 year were genotyped. The polymorphisms considered are the BclI in the NR3C1 gene, C-511T in IL-1ß gene, and Leu155His and rs2670660/C in NALP1 gene. Patients were grouped as responder, dependant, and resistant to GCs. The relation between GC response and the genetic polymorphisms considered was examined using univariate, multivariate, and Classification and Regression Tree (CART) analysis. RESULTS: Univariate analysis showed that BclI polymorphism was more frequent in responders compared with dependant patients (P=0.03) and with the combined dependant and resistant groups (P=0.02). Moreover, the NALP1 Leu155His polymorphism was less frequent in the GC responsive group compared with resistant (P=0.0059) and nonresponder (P=0.02) groups. Multivariate analysis comparing responders and nonresponders confirmed an association between BclI mutated genotype and steroid response (P=0.030), and between NALP1 Leu155His mutant variant and nonresponders (P=0.033). An association between steroid response and male sex was also observed (P=0.034). In addition, Leu155His mutated genotype was associated with steroid resistance (P=0.034). Two CART analyses supported these findings by showing that BclI and Leu155His polymorphisms had the greatest effect on steroid response (permutation P value=0.046). The second CART analysis also identified age of disease onset and male sex as important variables affecting response. CONCLUSIONS: These results confirm that genetic and demographic factors may affect the response to GCs in young patients with IBD and strengthen the importance of studying high-order interactions for predicting response.

Carbamazepine hypersensitivity syndrome triggered by a human herpes virus reactivation in a genetically predisposed patient.

A case of severe hypersensitivity syndrome, triggered by carbamazepine in the presence of a concomitant active human herpes virus (HHV) 6 and 7 infection is described. To further understand the molecular mechanism of this adverse reaction, analyses of the genetic variants of human leukocyte antigen (HLA) and of the epoxide hydrolase gene (EPHX1), previously associated with carbamazepine hypersensitivity, were performed. A lymphocyte transformation test (LTT) was conducted in order to detect drug-specific lymphocytes. In the hypersensitive patient, 2 genetic factors previously associated with intolerance to carbamazepine were detected: the allele HLA-A*3101 and homozygosity for the variant allele of SNP rs1051740 in EPHX1. Drug-specific lymphocytes could be detected by LTT when the HHV was active (positive PCR for viral DNA and increased anti-HHV 6 IgG titer), but not when it was no longer active. In conclusion, we document a case of severe carbamazepine hypersensitivity triggered by viral reactivation in a patient presenting the interaction of 2 unfavorable genetic factors.

Response to glucocorticoids and toxicity in childhood acute lymphoblastic leukemia: role of polymorphisms of genes involved in glucocorticoid response.

BACKGROUND: Glucocorticoids (GCs) play a fundamental role in the treatment of pediatric acute lymphoblastic leukemia (ALL), but therapy with these agents often results in a number of severe side effects. The aim of our study was to evaluate the association between polymorphisms of genes encoding for proteins involved in the pharmacokinetics/pharmacodynamics of these drugs and the occurrence of side effects, in particular infections, in a small population of ALL children. PROCEDURE: Common polymorphisms of NR3C1, ABCB1, glutathione-S-transferase (GST)-M1, GST-P1, GST-T1, and IL-10 genes were analyzed in 36 pediatric patients with ALL, treated according to the AIEOP-BMF ALL 2000 study protocol. Toxicities occurring during the induction and reinduction periods were assessed and their association with genotypes was evaluated. RESULTS: In univariate analysis, the risk of severe infections was increased in subjects with the GST-M1 null genotype, while patients with the GST-M1 normal genotype had significantly more moderate degree infections. The results were confirmed by multivariate analysis. Selection from the reference models of independent variables based on Akaike Information Criteria (AIC) scores maintained the GST-M1 genotype variable in the model to predict severe infections, and the ABCB1 C3435T and GST-M1 genotype variables in the model for moderate infections. CONCLUSIONS: GST-M1 genotype may influence the severity of infections in ALL children during GC administration.

Glutathione-S-transferase-P1 I105V polymorphism and response to antenatal betamethasone in the prevention of respiratory distress syndrome.

PURPOSE: The aim of this pilot study was to assess the association between polymorphisms in genes that encode for proteins involved in the pharmacokinetics/pharmacodynamics of glucocorticoids and the occurrence of respiratory distress syndrome (RDS) in preterm infants born to mothers treated with a complete course of betamethasone. METHODS: Sixty-two preterm infants were enrolled. The C1236T, G2677T, and C3435T polymorphisms in the ABCB1 gene, BclI, N363S and ER22/23EK in the NR3C1 gene, I105V in the GST-P1 gene and GST-M1 and GST-T1 deletions were analyzed, and their association with the occurrence of RDS was assessed. RESULTS: In univariate analysis, the heterozygous and homozygous presence of the I105V variant in the GST-P1 gene seemed to confer protection against the occurrence of RDS (P = 0.032), while no association for all other polymorphisms was observed. In multivariate analysis, selection from the reference model of independent variables based on AIC (Akaike information criteria) maintained three variables in the model: gestation, C3435T, and GST-P1 genotype. CONCLUSIONS: Polymorphisms of the GST-P1 gene may influence the effect of antenatal steroids on the newborn lung.

ABCB1 gene polymorphisms and expression of P-glycoprotein and long-term prognosis in colorectal cancer.

BACKGROUND: P-glycoprotein (Pgp), encoded by the ATP-binding cassette B1 (ABCB1) gene, is an efflux transporter located on the luminal side of intestinal epithelial cells, which protects the gut from endogenous and exogenous toxins. The association of two ABCB1 polymorphisms with the occurrence of colon cancer and long-term prognosis was evaluated in a selected patient population. The expression of Pgp in neoplastic and normal intestinal mucosa was also studied. PATIENTS AND METHODS: Archival material from 51 patients, in Dukes stage B2 or C, treated for 6 months with 5-fluorouracil plus leucovorin was retrieved. The G2677T and C3435T polymorphisms were studied and immunohistochemical analysis of the tumor and adjacent normal tissue was performed. RESULTS: The distribution of wild-type and polymorphic genotypes was similar in the patients and controls and in the patients who relapsed and those who remained event-free for 5 years. Cox proportional hazard model indicated an increased probability of relapse for older patients (p = 0.042) and C stage tumors (p = 0.030). Pgp expression was significantly lower in cancer tissue compared to normal mucosa (p < 0.001) and was related to grading, being lower in poorly-differentiated tumors (p < 0.05); however, no relationship was seen between Pgp expression, genotype and long-term prognosis. CONCLUSION: G2677T and C3435T polymorphisms are not associated with colon cancer risk and prognosis in a selected patient population.

Role of ABC Transporters in the BeWo Trophoblast Cell Line.

ABSTRACT The transport of doxorubicin and rhodamine 123, substrates of ABC transporters, was evaluated in the BeWo stabilized trophoblast cell line. Both compounds were taken up by BeWo cells, but their intracellular concentrations were highly dependent on temperature, and significantly reduced at 4 degrees C. The P-glycoprotein inhibitors verapamil and PSC833 did not modify the intracellular concentrations of the two substrates, suggesting therefore that, in these cells, the activity of P-glycoprotein is not important. MK571, which inhibits MRPs, was on the contrary effective in increasing rhodamine 123 intracellular concentrations. The efflux of both fluorescent substrates was extremely slow, and slightly reduced by MK571. Finally, a polarized transport of doxorubicin from basal to apical side was evident, although only during the first 60 min of incubation, and was reduced by P-glycoprotein, MRP, and BCRP inhibitors. No MDR1 expression was revealed at the mRNA and protein levels; on the contrary, MRP1 and BCRP were expressed in these cells. In BeWo cells the activity of ABC transporters, and in particular of P-glycoprotein, seems to be extremely limited.

Glutathione-S-transferase genotypes and the adverse effects of azathioprine in young patients with inflammatory bowel disease.

BACKGROUND: Adverse drug reactions to azathioprine, the prodrug of 6-mercaptopurine, occur in 15%-38% of patients and the majority are not explained by thiopurine-S-methyltransferase (TPMT) deficiency. Azathioprine is known to induce glutathione depletion and consumption of glutathione is greater in cells with high glutathione-S-transferase (GST) activity compared with those with low activity; moreover, some reports indicate that GST might play a direct role in the reaction of glutathione with azathioprine. The association between polymorphisms of GST-M1, GST-P1, GST-T1, and TPMT genes and the adverse effects of azathioprine was therefore investigated. METHODS: Seventy patients with inflammatory bowel disease (IBD), treated with azathioprine, were enrolled and clinical data were retrospectively determined. TPMT and GST genotyping were performed by polymerase chain reaction (PCR) assays on DNA extracted from blood samples. RESULTS: Fifteen patients developed adverse effects (21.4%); there was a significant underrepresentation of the GST-M1 null genotype among patients developing adverse drug reactions to azathioprine (odds ratio [OR] = 0.18, 95% confidence interval [CI] = 0.037-0.72, P = 0.0072) compared with patients who did not develop adverse effects. Patients heterozygous for TPMT mutations presented a marginally significant increased probability of developing adverse effects (OR = 6.38, 95% CI = 0.66-84.1, P = 0.062). Moreover, among the 55 patients who did not develop adverse effects, there was a significant underrepresentation of the GST-M1 null genotype among patients who displayed lymphopenia as compared with those that did not display this effect of azathioprine (OR = 0.15, 95% CI = 0.013-1.08, P = 0.032). CONCLUSION: Patients with IBD with a wildtype GST-M1 genotype present increased probability of developing adverse effects and increased incidence of lymphopenia during azathioprine treatment.

Rifampicin and verapamil induce the expression of P-glycoprotein in vivo in Ehrlich ascites tumor cells.

The effect of an in vivo treatment with two commonly employed drugs that are P-glycoprotein substrates, verapamil and rifampicin, on Ehrlich ascites carcinoma cells, was evaluated. Ehrlich ascites carcinoma cells were inoculated i.p. in CD-1 mice and animals were orally treated for 10 days with rifampicin (60 mg/kg/day) or verapamil (6 mg/kg/day). In the harvested cells the transcripts for mdr1a and mrp1, but not those for mdr1b, mrp2 and CYP3A, were detected, and treatment with verapamil or rifampicin did not modify the levels of the transcripts. On the contrary, an increased expression of P-glycoprotein was observed at the protein level with Western blot. The intracellular uptake of doxorubicin, a P-glycoprotein and MRP substrate, was significantly lower in cells obtained from treated animals in comparison with cells obtained from controls; in addition, the uptake was increased by a pretreatment with verapamil. The survival time of control animals implanted with untreated cells was similar to that of animals inoculated with cells obtained from rifampicin treated animals, however, the antineoplastic effect of doxorubicin was significanly higher in control animals. A treatment with rifampicin or verapamil in Ehrlich ascites tumor confers resistance to the antineoplastic drug doxorubicin, probably through an increased expression of P-glycoprotein.

Induction of proteins involved in multidrug resistance (P-glycoprotein, MRP1, MRP2, LRP) and of CYP 3A4 by rifampicin in LLC-PK1 cells.

P-glycoprotein, multidrug resistance-related proteins (MRPs) and lung resistance-related protein (LRP) are involved in multidrug resistance in tumor cells but are also expressed in normal tissues. In the LLC-PK(1) tubular renal cell line, a 15-day treatment with 25 microM rifampicin significantly increased the mRNA levels of P-glycoprotein, MRP1, MRP2, LRP and cytochrome P450 3A4 (CYP 3A4). Western blot analysis confirmed a moderate increase in the expression of P-glycoprotein and MRP2, but not MRP1 also at the protein level. The intracellular uptake of doxorubicin was significantly lower in rifampicin pretreated cells. A pretreatment with 6-[82S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]cyclosporin D, valspodar (PSC 833), a specific inhibitor of P-glycoprotein, with (3-(3-(2-(7-chloro-2-quinidinyl)ethenyl-phenyl)((3-diimethyl amino-3oxo propyl)thio)methyl)thio)propanoic acid, sodium salt (MK-571), a specific inhibitor of MRP1, and with verapamil, that inhibits both proteins, significantly increased doxorubicin cell accumulation in rifampicin pretread cells. In rifampicin treated cells cultured on porous membranes, doxorubicin showed a polarized transport, that was reduced by a pretreatment with PSC 833. A chronic treatment with rifampicin induces the expression of transport proteins and of CYP 3A4 and could therefore alter the renal elimination kinetics of drugs that are their substrates.

Toxicologic and pharmacokinetic study of low doses of verapamil combined with doxorubicin.

The effect of a chronic treatment with low oral doses of verapamil, a calcium channel blocker commonly employed in cardiovascular therapy, on doxorubicin toxicity, was evaluated in CD1 mice. Verapamil, administered at a dosage corresponding to a typical cardiovascular posology in humans, significantly increased doxorubicin toxicity. In particular the mortality was significantly higher and earlier and histological analysis revealed an increase in the severity of lesions in the liver, kidney and small bowel of verapamil pretreated animals. The pharmacokinetic profiles revealed that verapamil treated group had higher doxorubicin peak plasma and tissue levels and AUCs. This study shows that verapamil, administered at low doses, dramatically increases doxorubicin toxicity, probably through an interaction between the two drugs, both P-glycoprotein substrates, on the protein expressed in normal tissues, and suggests caution in the use of the calcium channel blocker for cardiovascular pathologies in patients who have to be treated with antineoplastic agents, substrates of P-glycoprotein.

Effect of periodontal therapy on the course of cyclosporin-induced gingival overgrowth: role of ABCB1 and PAI-1 gene polymorphisms.

OBJECTIVES: Etiological periodontal therapy is effective in reducing cyclosporin A-induced gingival overgrowth, but a high variability among subjects has been observed. This study aimed to evaluate the role of polymorphisms in PAI-1 and A BCB1 genes on the course of this side effect following periodontal therapy. METHOD AND MATERIALS: Forty-five transplant patients were subjected to nonsurgical periodontal therapy and evaluated for hypertrophy index, probing depths, bleeding, and plaque scores at baseline, and after 3 and 6 months. A BCB1 (C3435T and G2677T) and PAI-1 (4G/5G) polymorphisms were studied with polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction respectively. RESULTS: All the monitored periodontal indexes decreased significantly during the six months. Modeling of hypertrophy index by linearmixed- effect models (allowing non-normal distribution of the outcome variable hypertrophy index) resulted in the selection as the most significant model, of the one comprising the independent variables: time, C 3435T genotype, and their interaction term. This model indicated that C 3435T-mutated patients had significantly higher baseline hypertrophy index values (90% Markov chain Monte C arlo empirical confidence intervals: 5.08, 30.00). The decrease in hypertrophy index values over time showed a trend toward being faster in mutated than nonmutated patients (interaction time: C 3435T nonmutated, 90% Markov chain Monte C arlo empirical confidence interval: -11.08, -0.40). When hypertrophy index values were normalized, the significance and trend were lost. No effect of the A BCB1 G2677T and PAI-1 4G/5G polymorphisms was observed. CONCLUSION: These preliminary results suggest that C 3435T polymorphism is a genetic factor that could influence the course of cyclosporin A-induced gingival overgrowth in transplant patients subjected to periodontal therapy.

Usefulness of the measurement of azathioprine metabolites in the assessment of non-adherence.

Azathioprine is a thiopurine immunosuppressive antimetabolite used to chronically treat inflammatory bowel disease and autoimmune hepatitis. Azathioprine treatment is a long-term therapy and therefore it is at risk for non-adherence, which is considered an important determinant of treatment inefficacy. Measurement of 6-thioguanine and 6-methylmercaptopurine nucleotides has been recently suggested as a screener for non-adherence detection. We describe four young patients in which non-adherence to azathioprine therapy was detected only through the measurement of drug metabolite concentrations, and the criterion for non-adherence was undetectable metabolite levels. After the identification of non-adherence, patients and their families were approached and the importance of a correct drug administration was thoroughly enlightened and discussed; this allowed obtaining a full remission in all subjects. Our observations support the use of undetectable metabolite levels as indicators of non-adherence to therapy in azathioprine treated patients. The additional level of medical supervision given by this assay allows getting a better adherence to medical treatment, which results in an improvement in the response to therapy; these benefits may justify the costs associated with the assay.

St John's wort modulation and developmental expression of multidrug transporters in the rat.

Extracts of St John's wort (SJW) (Hypericum perforatum) are a potent inducer of enzymes of the cytochrome P450 system and of the transport protein P-glycoprotein, and interactions with a range of commonly prescribed medications have been described. In addition, recent experimental data suggest that, this otherwise safe treatment, could have some side effects when consumed during pregnancy and lactation. The aim of this study was to investigate, in Wistar rats, the effect of a treatment with high doses of SJW extract (100 and 1000 mg/kg/day) administered prenatally and during breastfeeding, on the level of transcripts of mdr1a, mdr1b, mrp1, mrp2 and cyp3A2 genes. All transcripts were detected in the liver, and their level of expression increased from fetuses to adults. SJW administration, at both dosages, caused a significant decrease of the levels of mdr1a, mdr1b, mrp1 and mrp2 in the livers of fetuses, and an increase in the levels of mdr1a, mdr1b, mrp2 and cyp3A2 in the mothers. In the other organs examined, a physiological regulation during ontogenesis was also evident, but SJW administration did not modify the expression level of the considered transcripts. These data suggest that the administration of the extract together with drugs that are substrates of transport proteins could be particularly hazardous during pregnancy.

Osteonecrosis of the hip after short courses of oral and inhaled steroids in a child with an increased number of glucocorticoid receptors.

We report an unusual case of osteonecrosis of the femoral head associated with recurrent myopathy and bone abnormalities in a two-year-old girl, in whom symptoms occurred after a ten-day course of oral betamethasone for infectious wheezy bronchitis, and eventually recurred and were worsened by topical treatment; a hypersensitivity to glucocorticoids is hypothesised.

Prevalence of methylenetetrahydrofolate reductase polymorphisms in young patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD) has been related to mutations of methylenetetrahydrofolate reductase (MTHFR), a critical enzyme in the metabolism of folate and methionine, both of which are important factors in DNA methylation and synthesis. A mutated MTHFR genotype was associated with increased toxicity of methotrexate treatment. The objective of this study was to verify, in a population of young patients with IBD, the presence of an association among mutations in the MTHFR gene, the incidence of IBD, and the risk of adverse events during the treatment with thiopurines azathioprine (AZA) or 6-mercaptopurine (6MP). Ninety-two patients with IBD were enrolled; 63 were treated with thiopurines; patients and 130 controls were genotyped for MTHFR mutations by PCR-based methods. The incidence of mutations in the MTHFR gene was not different between patients with IBD and control subjects; the mutated genotype was not associated with an increased risk of toxicity during thiopurine treatment.

Physiological regulation of P-glycoprotein, MRP1, MRP2 and cytochrome P450 3A2 during rat ontogeny.

P-glycoprotein and the multidrug resistance-related proteins MRP1 and MRP2 belong to the ATP binding cassette family of proteins and transport a wide range of substrates. These proteins are also involved in metabolic and excretory processes of xenobiotics. The rat genes mdr1a and mdr1b code for P-glycoproteins, while mrp1 and mrp2 genes code for MRP1 and MRP2 proteins, respectively. In this study, the physiological modulation of the level of transcript for these genes during rat ontogeny in the liver, kidney, lung, brain and heart was analyzed by reverse transcription-polymerase chain reaction. An increasing level of transcript during ontogeny was demonstrated for mdr1a and mdr1b in all tissues considered, as well as for mrp2, which was detected only in the liver and kidney. In contrast, mrp1 transcript, present in all tissues, did not show any modulation. The maximum level of expression was reached in adult animals and a significant decrease was demonstrated in aging rats. Western blot analysis with C219 and M2III-6 monoclonal antibodies confirmed this different pattern of expression during ontogeny in the liver. The physiological regulation of cytochrome P450 3A2 was also considered: in the rat liver, an increase in the level of transcript during ontogeny, with a maximum in 60-day-old rats and a decrease in 8-month-old rats, was evident.

Toxicity of Hypericum perforatum (St. John's wort) administered during pregnancy and lactation in rats.

The popularity of St. John's wort (Hypericum perforatum) for the treatment of depression is increasing and, in recent years, concerns about its use during pregnancy and breastfeeding have emerged. The purpose of this study was to investigate, in Wistar rats, the effects of a treatment with hypericum administered prenatally and during breastfeeding (from 2 weeks before mating to 21 days after delivery). Two doses of the extract were chosen, 100 mg/kg per day, which, based on surface area, is comparable to the dose administered to humans, and 1000 mg/kg per day. A microscopical analysis of livers, kidneys, hearts, lungs, brains, and small bowels was performed. A severe damage was observed in the livers and kidneys of animals euthanized postnatally on days 0 and 21. The lesions were more severe with the higher dose and in animals that were breastfed for 21 days; however, an important renal and hepatic damage was evident also with the dose of 100 mg/kg per day. In addition, similar serious hepatic and renal lesions were evident also in animals that were exposed to hypericum only during breastfeeding. In particular, a focal hepatic damage, with vacuolization, lobular fibrosis, and disorganization of hepatic arrays was evident; in the kidney, a reduction in glomerular size, disappearance of Bowman's space, and hyaline tubular degeneration were found. The results obtained in this study indicate that further, appropriate histological studies should be performed in other animal species to better evaluate the safety of hypericum extracts taken during pregnancy and breastfeeding.