Comparative Bioinformatic Analysis of the Proteomes of Rabbit and Human Sex Chromosomes.

Studying proteins associated with sex chromosomes can provide insights into sex-specific proteins. Membrane proteins accessible through the cell surface may serve as excellent targets for diagnostic, therapeutic, or even technological purposes, such as sperm sexing technologies. In this context, proteins encoded by sex chromosomes have the potential to become targets for X- or Y-chromosome-bearing spermatozoa. Due to the limited availability of proteomic studies on rabbit spermatozoa and poorly annotated databases for rabbits compared to humans, a bioinformatic analysis of the available rabbit X chromosome proteome (RX), as well as the human X (HX) and Y (HY) chromosomes proteome, was conducted to identify potential targets that could be accessible from the cell surface and predict which of the potential targets identified in humans might also exist in rabbits. We identified 100, 211, and 3 proteins associated with the plasma membrane or cell surface for RX, HX, and HY, respectively, of which 61, 132, and 3 proteins exhibit potential as targets as they were predicted to be accessible from the cell surface. Cross-referencing the potential HX targets with the rabbit proteome revealed an additional 60 proteins with the potential to be RX targets, resulting in a total of 121 potential RX targets. In addition, at least 53 possible common HX and RX targets have been previously identified in human spermatozoa, emphasizing their potential as targets of X-chromosome-bearing spermatozoa. Further proteomic studies on rabbit sperm will be essential to identify and validate the usefulness of these proteins for application in rabbit sperm sorting techniques as targets of X-chromosome-bearing spermatozoa.

Sustainable animal production: exploring the benefits of sperm sexing technologies in addressing critical industry challenges.

The sex of the animals is of paramount importance in many animal production systems. This is particularly evident in the production of milk or in breeding programs focused on the production of female animals. In some cases, slaughter or euthanasia of animals of the unwanted sex becomes the only solution, highlighting ethical and economic concerns. As global demand for food continues to rise, the importance of addressing these issues becomes more evident. Reproductive technologies, such as sperm sexing techniques, may hold the key to addressing both animal welfare and the sustainability of animal production. The use of semen enriched with sperm capable of producing offspring of the desired sex can serve as a valuable tool for producers to exert greater control over production outcomes, not only helping to mitigate welfare issues related to the unnecessary premature death of unwanted offspring but also providing a possible ally in the face of stricter animal welfare guidelines. In addition, sexed semen can also contribute to financial gains and reduce greenhouse gas emissions and food waste associated with the less profitable part of the herd. This paper explores the positive impacts that sperm sexing can have on animal welfare, economy, and environment. It also discusses currently available options and strategies for more successful implementation of sexed semen. Partnerships between companies and scientists will be essential to find innovative ways to adapt current production systems and develop sperm sexing technologies that apply to most livestock industries.

Pregnancy Complications and Feto-Maternal Monitoring in Rabbits.

Rabbit production holds significant relevance in modern agriculture due to its potential as a sustainable source of high-quality protein and efficient feed conversion, contributing to food security and economic diversification. Nevertheless, studies incorporating feto-maternal monitoring in this species are uncommon. This review gathers research on the monitoring and evaluation of factors affecting rabbit gestation, providing a better understanding of the causes of prenatal development abnormalities. These include studies regarding how chronic maternal hypertension, gestational diabetes, maternal stress, ectopic gestation, maternal uterine ischemia and fetal hypoxia, intrauterine growth restriction, superfetation, maternal age, maternal nutritional status, maternal physical condition, maternal and embryonic genotype, and the intrauterine location of rabbit fetuses can potentially impact rabbits' reproduction and maternal and fetal health. Among other monitoring techniques, ultrasonography, considered one of the best tools for diagnosing pregnancy and conducting follow-up, is also reviewed. Details on measurable fetal-development parameters in rabbits and precautions to be considered before and during the examination are also provided. Additional studies are required to understand why some events occur and their consequences throughout gestation, allowing the determination of new biomarkers or cut-offs that can be helpful for early diagnosis and improve reproductive efficiency.

The History and Prospects of Rabbit Sperm Sexing.

Sperm sex selection is a longstanding challenge in the field of animal reproduction. The cuniculture industry, in particular producers of males or females for breeding purposes, would greatly benefit from the pre-selection of the offspring's sex. This review article overviews the current and future developments in rabbit sperm sexing technologies, as well as the implications of implementing these methodologies in cuniculture. The first attempts of sperm sexing were performed in rabbits; however, a both efficient and cost-effective methodology was not yet developed for this species. Those included sperm sexing according to differences in sperm density, surface electric charge, pH susceptibility, antisera reaction, and flow cytometry. Separation by flow cytometry has proven to be efficient in rabbits, yielding fractions with approximately 81% and 86% purity for X- and Y-sperm, respectively. However, it is not cost-effective for cuniculture and decreases sperm quality. The advantages, limitations, and practical considerations of each method are presented, highlighting their applicability and efficiency. Furthermore, herein we explore the potential of immunological-based techniques that overcome some of the limitations of earlier methods, as well as recent advancements in sperm sexing technologies in other animal models, which could be applied to rabbits. Finally, the challenges associated with the development and widespread implementation of rabbit sperm sexing technologies are addressed. By understanding the advantages and limitations of existing and emerging methods, researchers can direct their efforts towards the most promising directions, ultimately contributing to a more efficient, profitable, and sustainable cuniculture.

Familiar del3p syndrome: The uncertainty of the prognosis. A case report.

The 3p deletion syndrome is an unusual condition. The few cases described are mainly de novo. We described a familial case detected in a prenatal diagnosis. Three members of the family had the 3p26.3-p26.1 deletion; however, only the son presented clinical features.

Sperm DNA damage and seminal antioxidant activity in subfertile men.

Supraphysiological ROS levels can lead to apoptosis, lipid peroxidation, and DNA and protein damage. This pilot study aimed to investigate the sperm oxidative damage in subfertile men, to describe the relationship between the antioxidant system and ROS. Sixty-four semen samples were categorised according to the evaluated routine parameters (WHO, WHO laboratory manual for the examination and processing of human semen, 2010). Results were cross-referenced with the DNA damage [Comet (n = 53) and TUNEL (n = 49) assays], antioxidant enzyme activity [SOD (n = 51), CAT (n = 48) and GST (n = 48)], and content of total thiols (n = 36), lipid hydroperoxides (n = 35) and MDA (n = 31). Compared to pathospermic samples, normozoospermic presented 40%-45% fewer spermatozoa with fragmented DNA, 19% fewer hydroperoxides, and slightly higher total thiols and MDA levels. Asthenozoospermic/asthenoteratozoospermic samples had the lowest GST activity. SOD and CAT showed a similar trend. Our results evidenced significant positive correlations between DNA damage and immotile spermatozoa; SOD and CAT, GST and total thiols; CAT and GST; total thiols and sperm concentration; and MDA levels and head/midpiece abnormalities and hydroperoxides. This work contributes to the existing body of knowledge by showing that the oxidative status correlates with the classic sperm analysis parameters. Oxidative stress and DNA damage evaluation might be a valuable diagnostic and prognostic tool in cases of idiopathic male subfertility.

Association of lifestyle factors with semen quality: A pilot study conducted in men from the Portuguese Trás-os-Montes and Alto Douro region followed in fertility support consultations.

A cross-sectional pilot study was conducted in men followed in fertility consultations, from the portuguese Trás-os-Montes and Alto Douro region, in order to associate several lifestyle factors with the spermatic parameters. Of a total of 522 men, 373 were compared based on the occupational exposure to harmful factors, smoking habits and practice of physical exercise per week, and the other 149 men according to their body mass index (normal weight vs. overweight vs. obesity). In the absence of harmful occupational factors, physical exercise seems to be associated with sperm quality improvement, whether individuals smoke or not. When exposed to harmful environments, non-smokers that practice physical exercise more than two times per week tended to present the best vitality, normal morphology and sperm concentration (p > .05). However, if they smoke, physical exercise seems not enough to enhance the spermatic parameters. The BMI correlated negatively with the spermatic quality, especially with sperm concentration (p < .05). Concerning men that did not present lifestyle risks associated, the motility, midpiece and tail abnormalities, and teratozoospermia index were significantly worse on obese individuals comparing to overweight men (p < .05). Thus, patients should also be recommended to control their weight and to have a BMI under 30 kg/m2 .

Elucidating the mechanisms of action of parecoxib in the MG-63 osteosarcoma cell line.

Different types of tumors often present an overexpression of cyclooxygenase-2. The aim of this study was to evaluate the effects of parecoxib (NSAID, cyclooxygenase-2 selective inhibitor) in the behavior of the human osteosarcoma MG-63 cell line, concerning several biological features. Cells were exposed to several concentrations of parecoxib for 48 hours. Cell viability/proliferation, cyclooxygenase-2 expression, morphologic alterations, membrane integrity, cell cycle evaluation, cell death and genotoxicity were evaluated. When compared with untreated cells, parecoxib led to a marked decrease in cell viability/proliferation, in COX-2 expression and changes in cell morphology, in a concentration-dependent manner. Cell recuperation was observed after incubation with drug-free medium. Parecoxib exposure increased lactate dehydrogenase release, an arrest of the cell cycle at S-phase and G2/M-phase, as well as growth of the sub-G0/G1-fraction and increased DNA damage. Parecoxib led to a slight increase of necrosis regulated cell death in treated cells, and an increase of autophagic vacuoles, in a concentration-dependent manner. In this study, parecoxib showed antitumor effects in the MG-63 human osteosarcoma cells. The potential mechanism was inhibiting cell proliferation and promoting necrosis. These results further suggested that parecoxib might be a potential candidate for in-vivo studies.

Synergistic Effect of Carboplatin and Piroxicam on Two Bladder Cancer Cell Lines.

BACKGROUND/AIM: This study aimed to evaluate the in vitro efficacy of carboplatin and piroxicam, both in isolation and combined, against T24 and 5637 human urinary bladder cancer cell lines. MATERIALS AND METHODS: Cell viability, drug interaction, cell morphology, cell proliferation, apoptosis and autophagy were analyzed after 72 h of drug exposure. Statistical analysis was performed and values of p<0.05 were considered statistically significant. RESULTS: Drug exposure in combination led to a significant reduction of cell viability comparatively to single-drug exposure. These combinations resulted in a synergistic interaction in the T24 (combination index for 50% effect (CI50)=0.65) and 5637 (CI50=0.17) cell lines. Notable increase of morphological alterations, a marked decrease of Ki-67 expression, a considerable enhancement of autophagic vacuoles and a minimal effect on apoptosis was observed in both cell lines treated with combined drugs. CONCLUSION: Data showed that in vitro combination of carboplatin and piroxicam produced a more potent antiproliferative effect when compared to single drugs.

Altered expression of CKs 14/20 is an early event in a rat model of multistep bladder carcinogenesis.

Cytokeratins (CKs) 14 and 20 are promising markers for diagnosing urothelial lesions and for studying their prognosis and histogenesis. This work aimed to study the immunohistochemical staining patterns of CK14/20 during multistep carcinogenesis leading to papillary bladder cancer in a rat model. Thirty female Fischer 344 rats were divided into three groups: group 1 (control); group 2, which received N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 20 weeks plus 1 week without treatment; and group 3, which received BBN for 20 weeks plus 8 weeks without treatment. Bladder lesions were classified histologically. CK14 and CK20 immunostaining was assessed according to its distribution and intensity. In control animals, 0-25% of basal cells and umbrella cells stained positive for CK14 and CK20 respectively. On groups 2 and 3, nodular hyperplastic lesions showed normal CK20 and moderately increased CK14 staining (26-50% of cells). Dysplasia, squamous metaplasia, papilloma, papillary tumours of low malignant potential and low- and high-grade papillary carcinomas showed increased CK14 and CK20 immunostaining in all epithelial layers. Altered CK14 and CK20 expression is an early event in urothelial carcinogenesis and is present in a wide spectrum of urothelial superficial neoplastic and preneoplastic lesions.

Effects of naproxen on cell proliferation and genotoxicity in MG-63 osteosarcoma cell line.

The purpose of this study was to determine the efficacy of naproxen, a nonsteroidal anti-inflammatory drug, on the MG-63 human osteosarcoma cell line. MG-63 cells were exposed to naproxen in a wide range of concentrations of 0.03, 0.05, 0.1, 0.42, 0.83, and 1.67 mg/ml for 72 h. The activity of naproxen was assessed by the following assays: cell morphology; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay; comet assay; and acridine orange and monodansylcadaverine (MDC) staining. Naproxen exerted a significant inhibitory effect on MG-63 cell proliferation, in a concentration-dependent manner, in all treatment groups compared with untreated cells. An increase in frequency of DNA damage, apoptotic bodies, apoptotic cells, and autophagic vacuoles was observed in MG-63-treated cells. Although future studies are needed, these findings suggest that naproxen may lead to improvements in treatment of patients with osteosarcoma.

Genomic characterization of three urinary bladder cancer cell lines: understanding genomic types of urinary bladder cancer.

Several genomic regions are frequently altered and associated with the type, stage and progression of urinary bladder cancer (UBC). We present the characterization of 5637, T24 and HT1376 UBC cell lines by karyotyping, fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MLPA) analysis. Some cytogenetic anomalies present in UBC were found in the three cell lines, such as chromosome 20 aneuploidy and the loss of 9p21. Some gene loci losses (e.g. CDKN2A) and gains (e.g. HRAS, BCL2L1 and PTPN1) were coincident across all cell lines. Although some significant heterogeneity and complexity were detected between them, their genomic profiles exhibited a similar pattern to UBC. We suggest that 5637 and HT1376 represent the E2F3/RB1 pathway due to amplification of 6p22.3, concomitant with loss of one copy of RB1 and mutation of the remaining copy. The HT1376 presented a 10q deletion involving PTEN region and no alteration of PIK3CA region which, in combination with the inactivation of TP53, bears more invasive and metastatic properties than 5637. The T24 belongs to the alternative pathway of FGFR3/CCND1 by presenting mutated HRAS and over-represented CCND1. These cell lines cover the more frequent subtypes of UBC and are reliable models that can be used, as a group, in preclinical studies.

Temsirolimus improves cytotoxic efficacy of cisplatin and gemcitabine against urinary bladder cancer cell lines.

OBJECTIVES: To analyze the cytotoxic action of temsirolimus using 3 established human bladder cancer cell lines and to assess whether temsirolimus potentiates the anticancer activity of gemcitabine and cisplatin. METHODS: Temsirolimus (500, 1,000, 2,000, and 4,000 nM), in isolation, and combined with gemcitabine (100 nM) and cisplatin (2.5 µg/ml), was given to 5637, T24, and HT1376 bladder cancer cell lines. Cell proliferation, autophagy, early apoptosis, and cell cycle distribution were analyzed after a 72-hour period. The expression of mammalian target of rapamycin baseline, Akt, and their phosphorylated forms, before and after treatment with temsirolimus, was evaluated by immunoblotting. RESULTS: Temsirolimus slightly decreased the bladder cancer cell proliferation in all 3 cell lines. No significant differences in the expression of mammalian target of rapamycin, Akt, and their phosphorylated forms because of temsirolimus exposure were found in the 3 cell lines. As part of a combined regime along with gemcitabine, and especially with cisplatin, there was a more pronounced antiproliferative effect. This pattern of response was similar to the other parameters analyzed (increased autophagy and apoptosis). Also, in the combined regime, an enhanced cell cycle arrest in the G0/G1 phase was observed. The non-muscle invasive 5637 bladder cancer cell line was most sensitive to both combinations. CONCLUSIONS: Temsirolimus makes a moderate contribution in terms of cell proliferation, apoptosis, and autophagy. However, it does potentiate the activity of gemcitabine and particularly cisplatin. Therefore, cisplatin- or gemcitabine-based chemotherapy regimen used in combination with temsirolimus to treat bladder cancer represents a novel and valuable treatment option, which should be tested for future studies in urinary bladder xenograft models.

Synergistic effect between cisplatin and sunitinib malate on human urinary bladder-cancer cell lines.

The aim of this paper is to analyse sunitinib malate in vitro ability to enhance cisplatin cytotoxicity in T24, 5637, and HT1376 human urinary bladder-cancer cell lines. Cells were treated with cisplatin (3, 6, 13, and 18 µM) and sunitinib malate (1, 2, 4, 6, and 20 µM), either in isolation or combined, over the course of 72 hours. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, acridine orange, and monodansylcadaverine staining and flow cytometry were performed. The combination index (CI) was calculated based on the Chou and Talalay method. In isolation, cisplatin and sunitinib malate statistically (P < 0.05) decrease cell viability in all cell lines in a dose-dependent manner, with the presence of autophagic vacuoles. A cell cycle arrest in early S-phase and in G0/G1-phase was also found after exposure to cisplatin and sunitinib malate, in isolation, respectively. Treatment of urinary bladder-cancer cells with a combination of cisplatin and sunitinib malate showed a synergistic effect (CI < 1). Autophagy and apoptosis studies showed a greater incidence when the combined treatment was put into use. This hints at the possibility of a new combined therapeutic approach. If confirmed in vivo, this conjugation may provide a means of new perspectives in muscle-invasive urinary bladder cancer treatment.

Cytogenetic characterization of an N-butyl-N-(4-hydroxybutyl) nitrosamine-induced mouse papillary urothelial carcinoma.

Chemically-induced urinary bladder cancer in rodents has long been used as a reliable model to study the biopathology of urinary bladder neoplasia and to develop therapeutic strategies against human tumors. Knowledge of the genetic basis underlying carcinogenesis would greatly enhance usability and usefulness of this model for the purposes of comparative pathology. However, little is known about the cytogenetic characteristics of rodent urinary bladder tumors. Accordingly, pathological and negative control specimens were collected for cytogenetic evaluation, from an ongoing mouse urinary bladder N-butyl-N-(4-hydroxybutyl) nitrosamine-induced carcinogenesis study. Histopathological analysis characterized the pathological sample as a papillary urothelial carcinoma. Conventional cytogenetic analysis revealed the presence of 66.3 % tetraploid cells. Fluorescent in situ hybridization using chromosome paint probes allowed the detection of a reciprocal translocation involving chromosomes 4 and 14 (containing the murine homologues to human p16 and retinoblastoma tumor-suppressor genes) in 42 % of tetraploid cells. The control sample showed no histological or cytogenetic changes. CDKN2A and RB1 loss of heterozygosity is associated with human early and advanced urinary bladder cancer, respectively. Thus, the present data paves the way for further studies concerning the molecular mechanisms of urinary bladder carcinogenesis.

In vitro and in vivo experimental models as tools to investigate the efficacy of antineoplastic drugs on urinary bladder cancer.

Several drugs have shown in vitro and in vivo pharmacological activity against urinary bladder cancer. This review aims at compiling the different drugs evaluated in in vitro and in vivo models of urinary bladder cancer and to review the advantages and limitations of both types of models, as well as the different methodologies applied for evaluating antineoplastic drug activity.

Meloxicam in the treatment of in vitro and in vivo models of urinary bladder cancer.

To assess the efficacy of meloxicam, a non-steroidal anti-inflammatory drug (NSAID), on three human urinary bladder-cancer cell lines (HT1376, T24 and 5637) and on mice urinary bladder cancer chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The in vitro effects of meloxicam were assessed by optical microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method, flow cytometry and comet assay. In vivo, Hsd:ICR male mice were exposed to BBN in drinking water, over the course of 12 weeks. Subsequently, animals were treated with meloxicam by intraperitoneal route, for 6 consecutively weeks. Tumour development was evaluated by haematoxylin and eosin staining. Renal and hepatic functions, interleucin-6 (IL-6), C-reactive protein (CRP) and tumour necrosis factor (TNFα) were also evaluated. In vitro, meloxicam induced a significant (P<0.05) decrease of cell proliferation. A significant (P<0.05) cell cycle arrest on G0/G1 phase was also detected in all the cell lines, with a slight but significant increase of sub-G0/G1 fraction on T24 (P=0.006) and 5637 (P<0.001) cells. Also a significant (P<0.05) increase in DNA damage was found on meloxicam-treated cells. In vivo, the incidence of pre-neoplastic lesions induced by BBN was not affected by meloxicam treatment. However, although not statistically significant, the development of neoplastic lesions was inhibited by meloxicam treatment without significant alterations of renal or hepatic parameters. Meloxicam is effective on in vitro and in vivo models of urinary bladder cancer. These findings support that meloxicam deserves more attention on urinary bladder cancer study.

Everolimus combined with cisplatin has a potential role in treatment of urothelial bladder cancer.

Cisplatin (CDDP)-based chemotherapy is a commonly treatment for advanced urothelial carcinoma. However, episodes of cisplatin resistance have been referenced. Recently it has been reported that everolimus (RAD001) could have an important role to play in bladder-cancer treatment and that mTOR inhibitors may restore chemosensitivity in resistant tumours. The aim of this study was to assess RAD001 in vitro ability to enhance CDDP cytotoxicity in three human bladder-cancer cell lines. Over the course of 72h, the cells were exposed to different concentrations of CDDP and RAD001, isolated or combined. Treatment with CDDP statistically (P<0.05) decreased cell proliferation in cell lines in a dose-dependent manner. The anti-proliferative activity of CDDP used in combination with RAD001 was statistically significant (P<0.05) in the cell lines at all concentrations tested. RAD001 had a therapeutic effect when used in combination with CDDP and could therefore be a useful anti-cancer drug combination for patients with bladder cancer.

In vivo and in vitro effects of RAD001 on bladder cancer.

OBJECTIVE: To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines. METHODS: ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms. RESULTS: The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G0/G1 phase arrest, as well as a statistically significant induction of apoptosis (P = 0.001), was only observed in the 5637 cell line. CONCLUSION: RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.

Everolimus enhances gemcitabine-induced cytotoxicity in bladder-cancer cell lines.

The purpose of this study was to determine whether everolimus, a rapamycin derivative, might significantly enhance the cytotoxicity of gemcitabine, an antitumor drug, in two human bladder-cancer cell lines. Human bladder-cancer T24 and 5637 cells were incubated with gemcitabine and everolimus in a range of concentrations either alone or in combination for 72 h. Flow cytometry, comet assay, MTT method and optical microscopy were used to assess cell proliferation, cell cycle, DNA damage, and morphological alterations. Gemcitabine exerted an inhibitory effect on T24 and 5637 cell proliferation, in a concentration-dependent manner. Everolimus significantly reduced proliferation of 5637 bladder cancer cells (IC30) at 1 µM), whereas T24 demonstrated marked resistance to everolimus treatment. A significant antiproliferative effect was obtained combining gemcitabine (100 nM) with everolimus (0.05-2 µM) with an arrest of cell cycle at S phase. Furthermore, an increase in frequency of DNA damage, apoptotic bodies, and apoptotic cells was observed when T24 and 5637 cancer cells were treated simultaneously with both drugs. Data show that in vitro combination produced a more potent antiproliferative effect when compared with single drugs.