Research protocol: Cisplatin-associated ototoxicity amongst patients receiving cancer chemotherapy and the feasibility of an audiological monitoring program.

BACKGROUND: Cisplatin is an anti-cancer chemotherapy drug classified as an alkylating agent. It is used for the treatment of a variety of cancers such as cervical, breast, stomach, prostate, bladder and oesophageal, to name a few. However due to its expansive toxicity profile, patients receiving cisplatin can experience high frequency hearing loss, a side effect known as ototoxicity. The dearth of information on the extent and severity of cisplatin-associated ototoxicity in South Africa prevents the implementation of a context-specific audiological monitoring programme. METHODS: This study aims to determine the extent and severity of ototoxicity amongst patients with cervical cancer, receiving cisplatin-based chemotherapy and hence the feasibility of an ototoxicity monitoring program in the province of KwaZulu-Natal, South Africa. A concurrent mixed methods design will be employed in the study. This longitudinal study will involve interviewing oncology nurses, oncologists, pharmacists and audiologists to assess the level of awareness to ototoxicity, as well as conducting diagnostic audiological evaluations at regular intervals on 78 patients with cervical cancer to ascertain the progression of hearing loss during and after chemotherapy. The feasibility of the monitoring program will be assessed as a parallel process to the audiological evaluations, where patient outcomes and cost implications to the patient and the health sector will be considered. Data will be subjected to statistical analyses so as to strengthen knowledge in the field and inform appropriate policies, and healthcare providers. DISCUSSION: This study is the first longitudinal study in South Africa to determine the ototoxic effects of cisplatin therapy on patients diagnosed with cervical cancer. Thus, the results generated from this study is likely to bring novel information to the fore using an evidence-based approach that will influence policy and clinical practice which can vastly improve the quality of life of patients undergoing chemotherapy. Mitigation of any further loss in the quality of life of affected patients is of paramount importance and the data generated from this project can lay the basis for further effective dialogue towards policy formulation on an ototoxic monitoring programme and the resultant strengthening of health systems in limited resource settings.

An overview of cancer research in South African academic and research institutions, 2013 - 2014

BACKGROUND AND OBJECTIVES:Cancer is emerging as a critical public health problem in South Africa (SA). Recognising the importance of research in addressing the cancer burden; the Ministerial Advisory Committee on the Prevention and Control of Cancer (MACC) research working group undertook a review of the current cancer research landscape in SA and related this to the cancer burden.METHODS:Academic and research institutions in SA were contacted to provide information on the titles of all current and recently completed (2013/2014) cancer research projects. Three MACC research working group members used the project titles to independently classify the projects by type of research (basic; clinical and public health - projects could be classified in more than one category) and disease site. A more detailed classification of projects addressing the five most common cancers diagnosed in males and females in SA was conducted using an adapted Common Scientific Outline (CSO) categorisation.RESULTS:Information was available on 556 cancer research projects. Overall; 301 projects were classified as clinical; 254 as basic science and 71 as public health research. The most common cancers being researched were cancers of the breast (n=95 projects) and cervix (n=43); leukaemia (n=36); non-Hodgkin's lymphoma (n=35) and lung cancer (n=23). Classification of the five most common cancers in males and females in SA; using the adapted CSO categories; showed that the majority of projects related to treatment; with relatively few projects on prevention; survivorship and patient perspectives.CONCLUSION:Our findings established that there is a dearth of public health cancer research in SA

Occurrence of the C-series fumonisins in maize from the former Transkei region of South Africa.

Fumonisins are a group of structurally related mycotoxins produced mainly in maize by Fusarium verticillioides and F. proliferatum. The most abundant naturally occurring analogue is fumonisin B(1) (FB(1)), with lesser amounts of fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) occurring. The C-series fumonisins (FCs) are structurally analogous to the B-series but lack the C-1 methyl group. Good and mouldy subsistence-grown maize samples were collected from the Centane and Bizana districts in the former Transkei region of South Africa. After extraction with methanol/water and clean-up on strong anion exchange solid phase extraction cartridges, FB(1), FB(2), FB(3), FC(1), FC(3) and FC(4) were determined by reversed-phase LC-MS/MS using positive ion electrospray ionisation. FB(1) levels in both good and mouldy maize from Centane (means (±SD) 2.75 ± 2.24 and 23.4 ± 12.5 mg kg(-1), respectively) were higher than the corresponding levels in maize samples from Bizana (means 0.056 ± 0.157 and 3.71 ± 5.01 mg kg(-1), respectively). Similarly, FC(1) levels in both good and mouldy maize from Centane (means 0.107 ± 0.099 and 0.814 ± 0.391 mg kg(-1), respectively) were higher than in Bizana, where FC(1) was detected in only one (0.018 mg kg(-1)) of 19 good maize samples and occurred in mouldy maize with a mean of 0.102 ± 0.135 mg kg(-1). A significant correlation (r=0.982, p<0.01) was observed between FB(1) and FC(1) levels in all samples, with FC(1) levels at 3.3% of the corresponding FB(1) levels. FC(4) levels were similar to FC(1), whereas only low amounts of FC(3) were detected.

Biomarkers of exposure to fumonisin mycotoxins: a review.

The investigation of adverse health effects associated with fungal mycotoxins requires the measurement of human exposure. Most frequently, this exposure is estimated from contamination levels of raw foodstuffs, which are the primary source of toxin exposure, and data on food consumption patterns. However, variations in food preparation methods, food intake, contamination level, intestinal absorption, toxin distribution and excretion lead to individual variations in toxin exposure that are more readily measured with a biomarker. Fumonisin biomarkers have been sought in the measurement of levels of the toxin in physiological samples such as serum, urine, faeces, hair and nails. However, due to the low bioavailability of fumonisin, these samples pose a variety of analytical challenges and also still require validation as biomarkers. The most widely researched fumonisin biomarkers have been those related to the disruption of de novo sphingolipid biosynthesis, namely elevated levels of the sphingoid base, sphinganine, or of its ratio with sphingosine. Elevation of these parameters in humans would potentially provide a biomarker of biochemical effect. A number of investigations into the possible elevation of sphinganine (or its ratio with sphingosine) in human blood and urine have generally failed to correlate with estimates of fumonisin exposure. The sphingoid bases occur naturally in human blood and urine such that their levels have normal ranges, which can be influenced by dietary factors other than fumonisin ingestion. The lower exposures from human diets, as compared with doses in experimental animals, have made detection of changes in these sphingoid biomarkers problematic.

An LC-MS-MS method for the determination of cyclizine in human serum.

Cyclizine is a piperazine derivative with anti-emetic activity that is useful in the prevention and treatment of nausea and vomiting associated with motion sickness. A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is presented for the quantitation of cyclizine in serum. Sample pretreatment involved liquid-liquid extraction of 200 microl of serum with dichloromethane after the addition of 100 microl each of ammonium hydroxide and internal standard solutions. The extracts were analyzed by HPLC on a Luna C18 reversed-phase column and an ion-trap mass spectrometer with an electrospray interface. A limit of detection of 1 ng/ml was determined which allowed for the reliable measurement of cyclizine in the serum of human subjects. The method was found to be linear over the calibration range of 2.5-100 ng/ml. The applicability of this method was demonstrated by the analysis of serum obtained from a human volunteer following administration of a single 50 mg cyclizine hydrochloride tablet. The reported method was observed to have the necessary sensitivity, selectivity, precision and accuracy for monitoring cyclizine concentrations in human subjects following oral administration.

The simultaneous determination of selected flavonol glycosides and aglycones in Ginkgo biloba oral dosage forms by high-performance liquid chromatography-electrospray ionisation-mass spectrometry.

An accurate, precise and sensitive liquid chromatography-tandem mass spectrometric (LC-MS-MS) method was developed for the determination of two flavonol glycosides, rutin and quercitrin, together with the algycone markers, quercetin, kaempferol and isorhamnetin in several Ginkgo biloba solid oral dosage forms. In addition, a novel quercetin glycoside, not yet reported in Ginkgo extracts, was identified. Liquid chromatography was performed using a minibore high-performance liquid chromatography (HPLC) column (150 mm x 2.0mm i.d.) and a one step gradient of acetonitrile-formic acid (0.3%) at a flow rate of 0.5 ml/min. Baseline separation of the five selected flavonol marker compounds was achieved within 20 min at 45 degrees C. Tandem mass spectrometry was performed using electrospray ionisation (ESI) in the negative ion mode. The marker compounds exhibited linearity over the range of 3-26 microg/ml and intra- and inter-day standard deviations were better than 7% and 16%, respectively. All Ginkgo products investigated were found to contain varying amounts of target analytes.

Golestan cohort study of oesophageal cancer: feasibility and first results.

To investigate the incidence of oesophageal cancer (EC) in the Golestan province of North-East Iran, we invited 1349 rural and urban inhabitants of Golestan province aged 35-80 to undergo extensive lifestyle interviews and to provide biological samples. The interview was repeated on a subset of 130 participants to assess reliability of questionnaire and medical information. Temperature at which tea was consumed was measured on two occasions by 110 subjects. Samples of rice, wheat and sorghum were tested for fumonisin contamination. An active follow-up was carried out after 6 and 12 months. A total of 1057 subjects (610 women and 447 men) participated in this feasibility study (78.4% participation rate). Cigarette smoking, opium and alcohol use were reported by 163 (13.8%), 93 (8.8%) and 39 (3.7%) subjects, respectively. Tobacco smoking was correlated with urinary cotinine (kappa = 0.74). Most questionnaire data had kappa > 0.7 in repeat measurements; tea temperature measurement was reliable (kappa = 0.71). No fumonisins were detected in the samples analysed. During the follow-up six subjects were lost (0.6%), two subjects developed EC (one dead, one alive); in all, 13 subjects died (with cause of death known for 11, 84.6%). Conducting a cohort study in Golestan is feasible with reliable information obtained for suspected risk factors; participants can be followed up for EC incidence and mortality.

Fumonisins in Brazilian corn-based foods for infant consumption.

A survey of 196 samples of corn-based infant foods from 13 cities of Sao Paulo State, Brazil, was carried out to investigate the fumonisin contamination in the products. Based on their ingredients, the products were divided into seven groups: infant cereal designated as types A-D, corn meal, corn starch and instant cereal baby food. Although certain infant food samples were free of fumonisin contamination (<20 microg kg(-1); corn starch and infant cereals of type A, B and D), contamination levels in the other products (corn meal, instant corn-based baby food and cereal type C) were of concern, particularly those in corn meal. All samples in these categories contained fumonisins. The mean level for total fumonisins (FB1 + FB2 + FB3) in corn meal was 2242 microg kg(-1) (maximum 8039 microg kg(-1)), in instant corn-based baby food was 437 (maximum 1096) microg kg(-1) and in infant cereal type C was 664 (maximum 1753) microg kg(-1).

Determination of the mycotoxin fumonisin B1 in maize by reversed-phase thin-layer chromatography: a collaborative study.

A simple and cost-effective method using thin-layer chromatography for the determination of the mycotoxin fumonisin B1 in maize is described. The analytical method consisted of the extraction of ground maize by shaking with methanol/water (75:25) for 60 min and clean-up of the resultant extract by means of strong anion exchange solid-phase extraction. The purified residue, formed by evaporation of the elution solvent, was reacted with fluorescamine and the fumonisin B1-derivative was separated by reversed-phase thin-layer chromatography using a developing solution of methanol/aqueous 4% potassium chloride (70:30). The derivatized FB1 was readily visualized as a greenish-yellow spot under long wavelength ultraviolet light and quantified by visual comparison with a set of similarly derivatized standards in the range 20-300 ng FB1 spotted on plate. Based on visual comparison, levels down to 0.5 mg kg(-1) were successfully estimated. The method was collaboratively studied in 14 laboratories using four duplicate maize meal samples (including a blank) and a spiked sample for determination of recovery. No significant difference was observed between mean FB1 levels by high-performance liquid chromatography or thin-layer chromatography. Based on within-laboratory relative standard deviations of 27.1-41.7% and between-laboratory relative standard deviations of 35.0-63.3%, the method can be considered semiquantitative. The mean recovery achieved by participants at a spiking level of 2.00 mg kg(-1) was 74.5%.

Assessing chronic exposure to fumonisin mycotoxins: the use of hair as a suitable noninvasive matrix.

This study describes for the first time the accumulation of measurable levels of fumonisin mycotoxins in the hair of nonhuman primates (vervet monkeys, Cercopithecus aethiops) and rats exposed to contaminated feed. Hair was subjected to reflux with methanol, and the resulting extract was cleaned up on strong anion exchange (SAX) and C18 solid-phase sorbents. Fumonisins FB1, FB2, and FB3 as well as their hydrolysis products commonly known as aminopolyols, AP1 and AP2, were detected in monkey hair using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS). Despite matrix interferences, the two-stage mass spectrometric process (MS-MS) yielded product ion mass spectra, which served as diagnostic indicators thus providing unequivocal identification of FB1, FB2, and FB3 as well as AP1 and AP2. In vervet monkeys, the levels of exposure related well to the levels of toxin detected in hair, and levels as high as 5.98 mg FB1, 33.77 mg FB1, and 65.93 mg FB1/kg of hair were found in monkeys receiving control, low-dose, and high-dose contaminated diets, respectively. Hair was also analyzed from rats given either single gavage doses of 1 and 10 mg FB1/kg body weight or contaminated feed (50 mg FB1/kg), resulting in an exposure of approximately 4.25 mg FB1/kg body weight/day based on the measured daily feed intake. Analysis of rat hair over a four-week period indicated that mean levels up to 34.50 mg/kg and 42.20 mg/kg were detectable by the fourth week in the rats treated by gavage (10 mg FB1/kg body weight) and those receiving contaminated feed, respectively. This relationship indicates that hair can provide an easily applicable non-invasive matrix for assessing chronic exposure to fumonisin mycotoxins.

Toxicokinetics of ochratoxin A in vervet monkeys (Cercopithecus aethiops).

The toxicokinetics of ochratoxin A were investigated in vervet monkeys (Cercopithecus aethiops). Three female monkeys were treated intravenously with ochratoxin A at doses, respectively, of 0.8, 1.5 and 2 mg/ kg body weight (BW). Blood and urine samples were collected over a period of 21 days. Plasma and urine extracts were analysed by high-performance liquid chromatography (HPLC) with either fluorescence or negative ion electrospray ionization mass spectrometric detection. The clearance of ochratoxin A from plasma followed a two-compartment model. The elimination half-life of ochratoxin A in the monkeys was determined to be 19-21 days and the average total body clearance was 0.22 +/- 0.07 ml/h per kg and the average apparent distribution volume of the central compartment was 59 +/- 9 ml/kg and the peripheral compartment was 59 +/- 20 ml/kg. No evidence was found for any metabolic conversion of ochratoxin A.

Supercritical fluid extraction and analysis of compounds from Clivia miniata for uterotonic activity.

In this descriptive study, the superciritical fluid extract of the roots of Clivia miniata L. was tested for uterotonic activity using guinea pig uterine smooth muscle in vitro. Extraction was performed with water modified supercritical carbon dioxide at 400 atm and 80 degrees C. The uterine contractions induced by this extract were compared to those induced by the aqueous extract and found to be active at lower doses. The active compounds were isolated and the structures elucidated by spectroscopic and chromatographic techniques. Both linoleic acid and 5-hydroxymethyl-2-furancarboxaldehyde isolated from the extract were found to induce muscle contractions individually. The pharmacological mode of action of 5-hydroxymethyl-2-furancarboxaldehyde was assessed using two receptor agonists and antagonists. This compound was found to mediate its effect through cholinergic receptors.

Determination of patulin in apple juice by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

An HPLC-MS-MS method with selected reaction monitoring (SRM) for the determination of patulin in apple juice samples is described. Mass spectrometric detection was accomplished following atmospheric pressure chemical ionization (APCI) in both positive and negative ion modes. Collision induced dissociation (CID) of the protonated molecular ion led initially to the loss of H2O (fragment m/z 137). At higher energies CO is lost from both the protonated parent molecule (fragment m/z 127) and the dehydrated molecular ion (fragment m/z 109). In contrast, CID of the deprotonated molecular ion led initially to the fragment at m/z 109 corresponding to the loss of either CO2 or acetaldehyde, followed at higher CID energy by the loss of H2O (fragment m/z 135) and CO (fragment m/z 125) from the deprotonated molecular ion. Detection in the negative ion mode proved superior and a linear response was observed over the injected range from 6 to 200 ng patulin. Apple juice samples spiked with patulin between 10 and 135 microg/l were analyzed following liquid-liquid extraction with ethyl acetate and clean up with sodium carbonate. Utilizing reversed-phase HPLC with acetonitrile-water (10:90) at 0.5 ml/min, levels down to 10 microg/l were readily quantified and a detection limit of 4 microg/l was attainable at a signal-to-noise (SIN) ratio of 4. The MS data for the spiked samples compared well to the UV data and when plotted against each other displayed a correlation coefficient (R) of 0.99.

The uterotonic activity of compounds isolated from the supercritical fluid extract of Ekebergia capensis.

The wood of Ekebergia capensis Sparrm. is used by the local Zulu community in KwaZulu-Natal Province, South Africa to facilitate childbirth. In this investigation, the uterotonic properties of extracts from this tree were evaluated using both pregnant and non-pregnant guinea pig uterine smooth muscle in vitro. The extracts were prepared using water modified supercritical carbon dioxide at 400 atm and 80 degrees C. As samples of these extracts displayed positive results when screened for uterotonic activity, gravity column chromatography followed by NMR spectroscopy was performed in an attempt to isolate and elucidate the structures of the compounds that were present in the extract. The extract yielded five known compounds of which only two, viz. oleanonic acid and 3-epioleanolic acid, displayed uterotonic activity. Receptor binding assays were subsequently performed with 3-epioleanolic acid to ascertain its mode of action. Bradykinin (30 ng/100 microl) and acetylcholine (1 microg/100 microl) were used as the B2 and cholinergic receptor agonists respectively with icatibant (HOE 140) (30 ng/100 microl) and atropine (60 micro/100 microl) as their corresponding antagonists. 3-epioleanolic acid was observed to mediate its effect through the cholinergic receptor. The results of this study show that two compounds from the extract of this tree possess varying degrees of agonist activity on uterine smooth muscle with minor changes in the molecular structure affecting its intrinsic activity on uterine muscle.

Production of the mycotoxins fusaproliferin and beauvericin by South African isolates in the Fusarium section Liseola.

The production of fusaproliferin (FUS), a recently described mycotoxin, and beauvericin (BEA), a mycotoxin recently reported to co-occur with FUS in Fusarium-infected corn, by South African isolates in the Fusarium section Liseola, was investigated. Five isolates each of F. verticillioides, F. proliferatum, F. subglutinans, and F. globosum were cultured on corn kernels. Four each of the five South African isolates of F. proliferatum and F. subglutinans produced FUS (10-1725 and 330-2630 mg/kg, respectively). BEA was produced by four of the F. proliferatum strains (310-1130 mg/kg) and three of the F. subglutinans strains (140-700 mg/kg). The isolates of F. verticillioides failed to produce significant levels of either of these secondary metabolites. F. globosum was a weak producer of both in that one isolate of five produced 25 mg/kg FUS and five out of five produced BEA at levels ranging between 10 and 110 mg/kg. To further characterize these strains, their production of fumonisins B(1), B(2), and B(3), as well as moniliformin, was investigated. Of the four species investigated, fumonisins were produced by all except F. subglutinans, which in turn was the only species whose isolates in this study produced moniliformin (four of five isolates, ranging from 155 to 2095 mg/kg). Analysis of visibly Fusarium-infected home-grown corn collected in the Transkei region of the Eastern Cape Province of South Africa showed that nine of the ten samples contained low levels of FUS (up to 62 microg/kg), whereas all ten samples showed BEA contamination ranging from 8 to 1734 microg/kg with a mean of 258 microg/kg.

Determination of the Fusarium mycotoxins, fusaproliferin and beauvericin by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A method is described using LC-MS for the detection of the mycotoxins fusaproliferin (FUS) and beauvericin (BEA) in cultures of Fusarium subglutinans and in naturally contaminated maize. Protonated molecular ion signals for FUS and BEA were observed at m/z 445 and m/z 784, respectively. Collision induced dissociation of the readily dehydrated protonated molecular ion of the sesterterpene FUS (m/z 427) led to the loss of another water molecule (m/z 409) and acetic acid (m/z 385), while the cyclic lactone trimer BEA fragmented to yield the protonated dimer (m/z 523) and monomer (m/z 262), respectively. Detection of FUS was best performed in the MS-MS mode while BEA displayed a stronger signal in the MS mode. The on-column instrumental detection limits for pure FUS and BEA were found to be 2 ng and 20 pg (S/N=2) while those in naturally contaminated maize were 1 microg/kg and 0.5 microg/kg, respectively. Five South African strains of F. subglutinans were analyzed following methanol extraction of which four produced FUS at levels between 330 mg/kg and 2630 mg/kg while only three produced BEA at levels between 140 mg/kg and 700 mg/kg. Application of this method to naturally contaminated maize samples from the Transkei region of South Africa showed FUS at levels of 8.8-39.6 microg/kg and BEA at 7.6-238.8 microg/kg.

Determination of the mycotoxin moniliformin in cultures of Fusarium subglutinans and in naturally contaminated maize by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A LC-MS method employing triethylamine as ion-pairing reagent for the determination of moniliformin in culture material and naturally contaminated maize samples is described. Mass spectrometric detection of moniliformin was accomplished following atmospheric pressure chemical ionization to yield the deprotonated molecular ion [M-H]- at m/z 97. The moniliformin response was found to be linear over the injected range 10 ng to 700 ng and a detection limit of 10 ng was attainable at a signal-to-noise (S/N) ratio of 4. Five South African strains of Fusarium subglutinans were grown on maize kernels and moniliformin extracted with an acetonitrile-water (95:5) mixture. Following sample clean up with reversed-phase (C18) solid-phase extraction cartridges, the extracts were subjected to LC-MS analysis. Triethylamine was used as an ion-pair reagent and found to improve the retention characteristics of moniliformin without any detrimental effects to the instrument. Moniliformin concentrations ranged between 130 mg/kg and 1460 mg/kg culture. Application of this method to naturally contaminated maize samples from Transkei showed that it was capable of measuring moniliformin levels down to 10 micrograms/kg in selected moldy maize cobs. This is the first report on the application of LC-MS to the analysis of moniliformin in cultures of F. subglutinans and in naturally contaminated maize.

Coupling SFE to uterotonic bioassay: an on-line approach to analysing medicinal plants.

Supercritical fluid extraction has been directly coupled on-line to a uterotonic bioassay, using guinea pig uterine smooth muscle in vitro. This technique was developed for the detection of uterotonic compounds present in medicinal plants used during pregnancy to induce or augment labour. The direct passage of CO2 into the muscle chamber led to adiabatic cooling of the physiological fluid and inhibition of muscle contraction. This was alleviated by the construction of a CO2 reduction interface together with the passage of carbogen which aided in the rapid displacement of excess CO2. The on-line system was evaluated with four plants (Clivia miniata (Lindl.) Regel, Ekebergia capensis Sparrm., Grewia occidentalis L. and Asclepias fruticosa L.) that are currently used during pregnancy by some black South African women. Extractions were performed with water modified supercritical CO2. Fractions of supercritical fluid extracts, obtained by sequentially increasing the pressure from 200 to 300 and 400 atm at constant temperature were transferred directly to the muscle chamber to identify the active fractions. The 400 atm extracts of C. miniata, A. fruticosa and E. capensis displayed maximum uterotonic activity while only the 300 atm extract of G. occidentalis induced uterine muscle contraction. This technique proved to be a safe and sensitive method for analyzing medicinal plants that contain uterotonic substances hence assisting in rapidly validating the uterotonic properties and detecting any toxic effects of these extracts.