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Antiproliferative activity of chloroformic fractions from leaves and inflorescences of Ageratina gracilis

Méndez-Callejas, Gina | 2017

To obtain a scientific basis and justification of plant domestication in the use of Ageratina gracilis, we did an in vitro study of the anticancer potential of extracts and fractions from its leaves and inflorescences. Firstly, cytotoxicity was evaluated against five human tumorigenic cell lines by MTT assay. Subsequently, the chloroformic fractions, considered the most cytotoxic were tested for genotoxicity by comet assay, morphological effects were analyzed by fluorescent microscopy, cell cycle arrest by flow cytometry and early apoptosis induction through fluorescein-5-isothiocyanate (FITC) labeled Annexin-V assay. Non-polar extracts with IC50 values of <53μg/ml showed a high cytotoxicity. The highest cytotoxicity was achieved by chloroformic fraction from petroleum ether extract of leaves and inflorescences and chloroformic fraction from ethanolic extract of leaves, displaying a significant inhibition of cell viability particularly on A549 cells with an IC50 value of 25.9 μg/mL. Chloroformic fractions caused a high percent of DNA damage above 60 percent on A549 and MDAMB-231.The fractions also induced G1/S phase arrest of the cell cycle in A549 cells, furthermore it was confirmed the apoptotic activity chloroformic fraction from petroleum ether extract of inflorescences and chloroformic fraction from ethanolic extract of leaves on those cells by Annexin-V assay. These preliminary results indicate that A. gracilis has an antiproliferative activity against cancer cells, being a starting point for forthcoming studies about the antineoplastic activity and its domestication conditions

Chemical and Biological Activity of Leaf Extracts of Chromolaena leivensis

Torrenegra G., Ruben D. | 2011

The flavonoids 3,5-dihydroxy-7-methoxy-flavanone, 3,5-dihydroxy-7-methoxyflavone and 3,5,7-trihydroxy-6-methoxyflavone were isolated from the leaves of C. leivensis. Preliminary observations in K562 cells (human erythroleukemia) using the trypan blue test, showed a 90% viability at a concentration of 100 µg/mL; however, further testing of the flavonoids at concentrations of 25, 50 and 100 µg/mL showed toxicity affecting the morphology of human erythroleukemia cells (K562) and human melanoma cells (A375). Induction of apoptosis was produced by 3.5-dihydroxy-7-methoxyflavone at 72 hours after treatment with arrest in the G2 / M phase of the cell cycle. The A375 cells treated with 50 µg/mL of 3.5-dihydroxy-7-methoxy-flavanone for 24, 48 and 72 hours, display effects on the behavior of the cell cycle. The flavonoid 3,5-dihydroxy-7-methoxyflavone has activity on the mitochondrial membrane at concentrations of 25, 50 and 100 µg/mL, at time intervals of 8 to 12 hours. The flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-dihydroxy-7-methoxyflavone at a concentration of 25 µg/mL increased the expression of costimulatory molecules corresponding to the phenotype presented by mature dendritic cells with differentiation markers CD40, CD83, CD86 and HLA-DR. The two flavonoids at concentrations between 0.39 and 100 µg/mL slightly increased the proliferation of peripheral blood mononuclear cells in the presence and in the absence of phytohemagglutinin. These flavonoids at concentrations of 50 and 100 µg/mL slightly increased the proliferation of fibroblasts.

Cinética de la actividad de las proteínas Bcl-2, BimEL Y Bax en apoptosis inducida por paclitaxel en la línea celular MDA-MB-231

González Saldarriaga, William Darley | 2017

Las células de cáncer de mama triple negativo se caracterizan por no poseer receptores para estrógenos, progesterona y HER2, lo que favorece la resistencia a quimioterapéuticos. Tal es el caso de la línea celular MDA-MB-231, que posee una elevada expresión de la proteína anti-apoptótica Bcl-2 afectando la activación de la vía intrínseca mitocondrial, necesaria para inducir el proceso de muerte celular programada. Por otra parte, no hay reportes de los niveles de expresión y la formación de complejos con las proteínas pro-apoptóticas BimEL y Bax durante el tratamiento con Paclitaxel, uno de los agentes anticancerígenos más utilizados en la actualidad. Se estableció el IC50 de Paclitaxel sobre MDA-MB-231 y se realizaron análisis con los extractos proteicos por medio de Inmuno-transferencia y co-inmunoprecipitación, identificando un aumento significativo tanto en la expresión como en la interacción entre las proteínas BimEL y Bax, mientras que la cantidad de Bcl-2 aumentó pero no significativamente y su grado de interacción con BimEL y Bax disminuye a medida que transcurre el tiempo de tratamiento con Paclitaxel. De acuerdo con los resultados, Paclitaxel induce apoptosis en MDA-MB-231 activando la formación de complejos que favorecen la permeabilidad de la membrana externa mitocondrial, de manera independiente a la sobre-expresión de Bcl-2.

Combretastatin A-4 induces p53 mitochondrial-relocalisation independent-apoptosis in non-small lung cancer cells

Méndez Callejas, Gina Marcela | 2014

CombretastatinA-4(CA-4)isoneofthemosteffectiveagentsusedinchemotherapy.Nevertheless,thecontributionofp53and Bim proteins in the CA-4-induced apoptosis in non-small lung cancer cells (NSCLC) remains unresolved, specifically on involving of p53 in the mitochondrial pathway activation by a transcription-independent mechanism. In this context, the p53null H1299 and wt-p53 H460 NSCLC cells, in the absence and presence of pifithrin-m (PFTm), an inhibitor of p53 mitochondrial-translocation, were treated with CA-4 and different cellular endpoints were analysed. In contrast to previous observationsinH460cells,CA-4failedintheactivationofanapoptoticresponseinH1299cells,thusindicatinganinvolvement of p53 in the cell death induced by the drug. We found that CA-4 led to p53 cellular re-localisation in H460 cells; in particular, p53 was released from the microtubular network and accumulated at mitochondria where it interacts with Bim protein and other proteins of the Bcl-2 (B-cell leukaemia-2) family, leading to cytochrome c release, alteration in the mitochondrial membrane polarisation, cell cycle arrest at the G2/M-phase, and cell death. Interestingly, the cytosolic and the mitochondrial accumulation of protein Bim was strictly dependent on p53 status. The extent of cell death was not reduced in H460 after combinedtreatmentofPFTmwithCA-4.Overall,thedatasupportamodelofCA-4-inducedapoptosisinNSCLC,forwhichthe expression of p53 protein is essential, but its mitochondrial function, linked to p53-transcription independent apoptosis pathway, is negligible.

Effects of BPA on expression of apoptotic genes and migration of ovine trophectoderm (oTr1) cells during the peri-implantation period of pregnancy

Elmetwally, Mohammed A. | 2019

Bisphenol A (BPA) is an endocrine-disrupting chemical used in the manufacture of many products used daily. In the present study, the effects of BPA (1 × 10 −4 to 1 × 10 -9 M) on migration and on the expression of some apoptotic genes were examined in vitro using ovine trophectoderm (oTr1) primary cell line. The results revealed that BPA at 1 × 10 −9 , 1 × 10 −8 and 1 × 10 −7 M increased migration of oTr1 cells, while 1 × 10 −6 , 1 × 10 −5 and 1 × 10 −4 M BPA decreased cell migration. Regarding apoptosis, expression of the anti-apoptotic gene Bcl-2 mRNA was greater at 1 × 10 −8 and 1 × 10 −9 M BPA and was down-regulated at 1 × 10 −4 to 1 × 10 −7 M BPA; however, expression of pro-apoptotic genes (Bax, cathepsin B, caspase-3 and c-myc) was reduced at the higher concentrations of BPA. Results of this study suggest that BPA may impair implantation by decreasing migration of oTr1 cells and inhibiting apoptosis.

Effects of catecholamines on secretion of interferon tau and expression of genes for synthesis of polyamines and apoptosis by ovine trophectoderm

Elmetwally, Mohammed A. | 2018

Ovine trophectoderm (oTr1) cells were used to investigate effects of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on their proliferation, migration and adhesion, secretion of interferon tau (IFNT), and expression of genes for synthesis of polyamines and apoptosis. Expression of mRNAs for agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1), and solute carrier family 7 (SLC7A1) (cationic amino acid transporter, Y + system), member 1 increased (P < 0.05) in oTr1 cells in response to EP and DA. However, expression of SLC7A1 decreased at high doses of EP and expression of ADC mRNA by oTr1 cells decreased in response to 20 and 40 ng/ml NE, and 40 ng/ml DA. Migration of oTr1 cells increased in response to EP, DA, and NE after 48 h of treatment. However, proliferation of oTr1 cells was inhibited by 300 pg/ml EP after 96 h and DA at 20 and 100 ng/ml. EP increased adhesion of oTr1 cells. The secretion of IFNT increased in response to 300 pg/ml EP, 100 ng/ml NE and DA after 48 h and at 96 h, and both DA (40 ng/ml) and NE (100 ng/ml). Expression of mRNAs for apoptotic genes (caspase 3, cathpsin B, BCL2 associated X protein “bax,” B-cell lymphoma 2 “bcl2,” and proto-oncogene “cmyc”) decreased (P < 0.05) in response to catecholamines, but DA did not affect (P < 0.05) expression of cMYC mRNA. These results indicate that catecholamines play important roles in conceptus development during the peri-implantation period of pregnancy through effects on synthesis of polyamines, secretion of IFNT, and expression of apoptotic genes by oTr1 cells.

Estudio computacional del acoplamiento molecular de la proteína P-53 con el flavonoide CT-1

Mahecha Moreno, Ingrith Zulay | 2022

El cáncer es una enfermedad que resulta de la interacción de un agente que lo causa y la reacción del organismo vivo; resulta crónica, pero es evitable y curable en un gran número de casos. Las teorías modernas de la carcinogénesis explican que se da por factores exógenos como agentes químicos, agentes físicos y agentes biológicos, también hay factores endógenos como el factor hereditario, el factor inmunitario, factor hormonal y factores nutritivos (Varela, 2002). Según la OMS el Cáncer es una de la segunda causa de muerte a nivel mundial, OMS (Organización Mundial de la Salud), sept. 2021), por esto ha venido siendo un campo muy estudiado, minimizando así dichos casos y convirtiéndolo en una enfermedad a la que se puede sobrevivir y otro porcentaje recuperarse en su totalidad. La funcionalidad de la proteína p-53 es principalmente un factor de transcripción y puede generar una variedad de programas anti-proliferativos al activar o reprimir genes efectores. El desarrollo de nuevos fármacos ha abierto paso a combinar la proteína p-53 con flavonoides, para potencializar su efecto anti-proliferativo. Las mutaciones en el gen p53 contribuyen aproximadamente la mitad de los casos de cáncer humano, ocurre cuándo se cambian la información del ADN en una posición y hacen que la célula construya p53 con un error, intercambiando un aminoácido incorrecto en un punto de la cadena de proteínas. En estos mutantes, la función normal de p53 está bloqueada y la proteína no puede detener la multiplicación en la célula dañada. (Goodsell, 2002). Los flavonoides pueden interferir en la carcinogénesis participando en el bloqueo o la supresión de diversos procesos, los efectos que se le atribuyen a los flavonoides son la capacidad de capturar radicales libres, así como la modificación de enzimas que desintoxican carcinógenos. El CT1 es un flavonoide con fórmula 3, 5, 4’- trihidroxi – 7 – metoxiflavona extraído a partir de la especie Chromolaena tacotana. En el presente trabajo se estudiaron las características del flavonoide CT-1 y de la proteína p-53, se reconocieron las interacciones moleculares del acoplamiento entre el p-53 y el flavonoide CT1, comparando dichas interacciones con el acoplamiento del flavonoide Quercetina y p-53.

Estudio teórico de acoplamiento molecular de Alquil benzoquinonas y Alqil fenoles con el dominio BIR3 de la proteina XIAP

Hernández Hernández, Michael Alexander | 2015

Los inhibidores de apoptosis (IAP) son un grupo de ocho proteinas (Apollon, cIAP1, cIAP2, XIAP, NAIP, Survivin, Livin y ILP-2) que poseen una funcion conservada evolutivamente para regular la muerte celular programada en las celulas animales desde insectos hasta los seres humanos. Dentro de la familia de las IAP se encuentra XIAP (X-linked inhibitor of apoptosis), proteina anti-apoptotica que es considerada como el inhibidor mas potente por su alta selectividad en la inhibicion de las caspasas 3, 7 y 9, que son las principales proteinas iniciadoras y ejecutoras de apoptosis. Naturalmente, XIAP es inhibida por la proteina mitocondrial Smac/DIABLO que tiene como funcion principal la regulacion de los inhibidores de apoptosis. Sin embargo, debido a la sobre expresion de la proteina anti-apoptotica XIAP en células de muchos tipos de cáncer, la cantidad de Smac liberado desde la mitocondria no es su ciente para controlar los niveles de XIAP evitando que la célula entre en proceso de muerte celular programada. Basado en la perturbacion del mecanismo de apoptosis debido a la sobre expresion XIAP, y en búsqueda de bloquear su capacidad inhibidora, en este estudio se evaluaron 16 moléculas que se distribuyen en dos moléculas con núcleo alquil benzoquinona y catorce con núcleo alquil fenol mediante la metodologia de docking molecular con el paquete computacional AutoDock 4.2, con el objetivo de determinar el complejo más estable que indique la inhibición de la proteina anti-apoptotica. En este estudio se hace uso de la metodologia de docking molecular debido a que permite hacer la prediccion de la conformacion preferida de una molecula (ligante) con respecto a una macromolecula de estructura conocida (receptor) y de esta forma determinar la estabilidad de este complejo. La optimizacion de geometria de las moléculas ligante se realizó usando el paquete computacional Gaussian 09 bajo la teoria de los funcionales de la densidad (DFT), al nivel de teor a B3LYP/6-31G. En este estudio, se obtuvo como resultado que las mol eculas MS4, MS6, MS10, MS11 y MS13 presentan la mayor a nidad con el receptor, siendo MS11 (-6,52 kcal/mol) el ligante que forma el complejo mas af n. Se encontr o que el ligante puede presentar a nidad con otros sitios sobre la super ficie de la proteina, por esta razon la localizacion inicial del ligante con respecto al receptor debe ser evaluada realizando variaciones de las coordenadas iniciales del grid para establecer el sitio activo con la mayor exactitud.xi

Interactome Analysis of Microtubule-targeting Agents Reveals Cytotoxicity Bases in Normal Cells

Gutiérrez Escobar, Andrés Julián | 2017

Cancer causes millions of deaths annually and microtubule-targeting agents (MTAs) are the most commonly-used anti-cancer drugs. However, the high toxicity of MTAs on normal cells raises great concern. Due to the non-selectivity of MTA targets, we analyzed the interaction network in a non-cancerous human cell. Subnetworks of fourteen MTAs were reconstructed and the merged network was compared against a randomized network to evaluate the functional richness. We found that 71.4% of the MTA interactome nodes are shared, which affects cellular processes such as apoptosis, cell differentiation, cell cycle control, stress response, and regulation of energy metabolism. Additionally, possible secondary targets were identified as client proteins of interphase microtubules. MTAs affect apoptosis signaling pathways by interacting with client proteins of interphase microtubules, suggesting that their primary targets are non-tumor cells. The paclitaxel and doxorubicin networks share essential topological axes, suggesting synergistic effects. This may explain the exacerbated toxicity observed when paclitaxel and doxorubicin are used in combination for cancer treatment.

Mechanism of Action of Two Flavone Isomers Targeting Cancer Cells with Varying Cell Differentiation Status

LeJeume, Timothy M. | 2015

Apoptosis can be triggered in two different ways, through the intrinsic or the extrinsic pathway. The intrinsic pathway is mediated by the mitochondria via the release of cytochrome C while the extrinsic pathway is prompted by death receptor signals and bypasses the mitochondria. These two pathways are closely related to cell proliferation and survival signaling cascades, which thereby constitute possible targets for cancer therapy. In previous studies we introduced two plant derived isomeric flavonoids, flavone A and flavone B which induce apoptosis in highly tumorigenic cancer cells of the breast, colon, pancreas, and the prostate. Flavone A displayed potent cytotoxic activity against more differentiated carcinomas of the colon (CaCo-2) and the pancreas (Panc28), whereas flavone B cytotoxic action is observed on poorly differentiated carcinomas of the colon (HCT 116) and pancreas (MIA PaCa). Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK) and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD) protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT.

A New Flavanone from Chromolaena tacotana (Klatt) R. M. King and H. Rob, Promotes Apoptosis in Human Breast Cancer Cells by Downregulating Antiapoptotic Proteins

Mendez, Gina | 2023

Chromolaena tacotana is a source of flavonoids with antiproliferative properties in human breast cancer cells, the most common neoplasm diagnosed in patients worldwide. Until now, the mechanisms of cell death related to the antiproliferative activity of its flavonoids have not been elucidated. In this study, a novel flavanone (3′,4′-dihydroxy-5,7-dimethoxy-flavanone) was isolated from the plant leaves and identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS). This molecule selectively inhibited cell proliferation of triple-negative human breast cancer cell lines MDA-MB-231 and MCF-7 whit IC50 values of 25.3 μg/mL and 20.8 μg/mL, respectively, determined by MTT assays with a selectivity index greater than 3. Early and late pro-apoptotic characteristics were observed by annexin-V/7-AAD detection, accompanied by a high percentage of the Bcl-2 anti-apoptotic protein inactivated and the activation of effector Caspase-3 and/or 7 in breast cancer cells. It was verified the decreasing of XIAP more than Bcl-2 anti-apoptotic proteins expression, as well as the XIAP/Caspase-7 and Bcl-2/Bax complexes dissociation after flavanone treatment. Docking and molecular modeling analysis between the flavanone and the antiapoptotic protein XIAP suggests that the natural compound inhibits XIAP by binding to the BIR3 domain of XIAP. In this case, we demonstrate that the new flavanone isolated from leaves of Chomolaena tacotana has a promising and selective anti-breast cancer potential that includes the induction of intrinsic apoptosis by downregulation of the anti-apoptotic proteins XIAP and Bcl-2. New studies should deepen these findings to demonstrate its potential as an anticancer agent.

Predicción de la actividad que tiene el péptido tma1 del veneno del escorpión Tityus Macrochirus sobre factores apoptóticos derivados de la familia de proteínas BCL-2

Andrade Cuervo, Beatriz Helena | 2022

En Colombia se pueden encontrar varias familias de escorpiones, una de estas es la familia Buthidae, dentro de esta familia pertenece el género Tityus, el cual cuenta con 29 especies; en donde se puede identificar el escorpión Tityus macrochirus. El veneno de este escorpión ha sido caracterizado a nivel bioquímico logrando identificar 4 péptidos dentro de los cuales se encuentra el Tma1 el cual es una toxina de mamíferos que puede afectar los subtipos de canales de Na+ humanos hNav1.6 y hNav1.4. En la actualidad se pueden encontrar varios estudios enfocados en el uso benéfico del veneno de los escorpiones. Se ha podido demostrar que el veneno de escorpión contiene propiedades anticancerígenas sobre varios tipos de cáncer lo cual puede inducir la apoptosis e inhibir varios mecanismos que promueven la proliferación y el crecimiento de las células cancerosas. Por medio de herramientas bioinformáticas como el acoplamiento molecular (docking) se desea predecir el mecanismo de acción y/o la actividad que tiene el péptido Tma1 del veneno del escorpión Tityus macrochirus sobre factores apoptóticos derivados de la familia de proteínas Bcl-2 para inducir la apoptosis ocasionando la inhibición del crecimiento de células cancerosas. Para ello, se lleva a cabo la identificación y caracterización a nivel primario, secundario y terciario del péptido Tma1, se identifica un inhibidor derivado de la familia de proteínas Bcl-2, se propone el mecanismo o actividad del péptido Tma1 en conjunto con el factor apoptótico derivado de la familia Bcl-2 seleccionado y finalmente se determinan las interacciones obtenidas del acoplamiento ligando-proteína

Semillero de Investigación e Innovación ORIATROS -2021 -II Docking molecular, flavonoides y apoptosis

Méndez Callejas, Gina Marcela | 2022-01-31

El Semillero de Investigación e Innovación ORIATOS, presenta las actividades realizadas en el periodo 2021-2, mencionando los resultados obtenidos, el estado de avance y las limitaciones en el desarrollo de su plan de trabajo

Semillero de Investigación e Innovación ORIATOS - 2021-II - Apoptosis actividad Anticancerígena

Méndez Callejas., Gina Marcela | 2022-01-31

El Semillero de Investigación e Innovación ORIATOS, presenta las actividades realizadas en el periodo 2021-2, mencionando los resultados obtenidos, el estado de avance y las limitaciones en el desarrollo de su plan de trabajo

Semillero de Investigación ORIATROS - Biología celular y molecular del cáncer: Informe de actividades 2020-II

Méndez Callejas, Gina Marcela | 2021-02-15

Semillero de Investigación ORIATROS - Biología celular y molecular del cáncer: Informe de actividades 2021-I

Méndez Callejas, Gina Marcela | 2021-06-23

The role of stem cells in uterine involution

Spooner, Madelyn | 2021

Uterine remodeling during pregnancy and repair postpartum are fundamental to the successful propagation of eutherian species. The most drastic remodeling occurs in species with invasively implanting embryos, including humans and mice. During embryo implantation, embryonic trophoblasts breach the epithelium, penetrating into the stroma. Stromal cell decidualization, which is critical for the establishment and maintenance of early pregnancy, occurs throughout the implantation site. Trophoblasts further invade into and remodel uterine spiral arteries, which is necessary for placental formation. The uterus increases in size up to 24-fold, which is largely attributed to myometrial expansion. Uterine changes that occur during pregnancy must then be resolved postpartum. Following parturition, the uterus repairs the remodeled tissue in the process of uterine involution. During involution, the majority of the endometrium is regenerated to replace the tissue that is shed postpartum. The myometrium returns to the pre-gravid state which is thought to occur through apoptosis and autophagy of smooth muscle cells. Although we understand the general process of postpartum uterine involution, the detailed mechanisms, particularly the role of putative stem cells, are poorly understood. This review discusses the evidence for the existence of epithelial, stromal and myometrial stem cells and their role in uterine involution. Gaps in knowledge and areas for future research are also considered. Studies of both postpartum and menstrual uterine repair, which likely involve similar mechanisms, are described under the broad definition of uterine involution. Although the primary focus of this review is human, mouse models are discussed to provide additional information.

Topological properties and in vitro identification of essential nodes of the Paclitaxel and Vincristine interactomes in PC-3 cells

Delgado Carreño, Claudia | 2019

Background: Microtubule-targeting agents (MTAs) disrupt microtubule dynamics, thereby inducing apoptosis via mitochondrial pathway activation through the modulation in the expression of the Bcl-2 family. Methods: To describe topological features of the MTAs networks associated to intrinsic apoptosis induction in p53-null prostate cancer cells, we predicted and compared the interactomes and topological properties of Paclitaxel and Vincristine, and thus, the essential nodes corresponding with the pro- and anti-apoptotic proteins and their kinetics were subjected to experimental analysis in PC-3 cell line. Results: The essential nodes of the apoptotic pathways, TP53, and CASP3, were identified in both, Paclitaxel and Vincristine networks, but the intrinsic pathway markers BCL2, BAX, and BCL2L1 were identified as hub nodes only in the Paclitaxel network. An in vitro analysis demonstrated an increase in BimEL and the cleaved-caspase-3 proteins in PC-3 cells exposed to both treatments. Immunoprecipitation analysis showed that treatments induced the releasing of Bax from the anti-apoptotic complex with Bcl-2 protein and the role of BimEL as a de-repressor from sequestering complexes, in addition, new protein complexes were identified between BimEL or Bcl-2 and cleaved-caspase-3, contributing data to the Vincristine network for p53-null cells in response to MTAs. Conclusion: The differences in sensitivities, protein profiles, and protein complex kinetics observed between the drugs confirmed that the selectivity and stimulation of the apoptotic system vary depending on the cell's genotype, the drug used and its exposure period.