Final Report: Proposal to Strengthen the EPSCoR Program for Environmental and Toxicological Research in Puerto Rico

EPA Grant Number: R827780E01
Title: Proposal to Strengthen the EPSCoR Program for Environmental and Toxicological Research in Puerto Rico
Investigators: Jimenez, Braulio D. , Bloom, Joseph , Cadilla, Carmen , Delgado, Wilfredo , Gonzalez, Fernando , Guadalupe, Ana R. , Rodriguez, Carlos , Santacana, Guido , Vega, Esther
Institution: University of Puerto Rico - Central Administration
EPA Project Officer: Hunt, Sherri
Project Period: October 1, 1999 through September 30, 2000
Project Amount: $499,999
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (1999) RFA Text |  Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)

Objective:

The overall objective of the University of Puerto Rico (UPR)-U.S. Environmental Protection Agency (EPA) Experimental Program to Stimulate Competitive Research (EPSCoR) was to strengthen the permanent presence of environmental-related research in Puerto Rico through the development of a productive environment for the achievement of national standards of excellence in environmental-related research. To achieve this objective, the Center for Environmental and Toxicological Research (CETR) coordinated and organized the expansion of two research projects in critical areas of environmental-related research. The two projects were to: (1) develop peptide nucleic acid biosensors for the detection of faecal coliforms in drinking water; and (2) research the toxicological effects of adsorbed compounds onto different size (total suspended particulates [TSP], particulate matter [PM2.5 and PM10]) of airborne PM from the Guaynabo Basin and a Reference Site (Fajardo).

Overall Achievements of the CETR

The CETR developed and undertook a research plan to evaluate the prevailing environmental conditions in an important component of the San Juan Bay Estuary System (SJBES). The SJBES consists of a group of bodies of water that includes the San Juan Bay, Condado, San José, Torrecilla, and Piñones Lagoons. This system is located on the northern coast of Puerto Rico. In 1992, the SJBES was declared by the EPA as an Estuary of National Importance that merits protection and environmental quality improvement. San José Lagoon (SJL), located in the 18° 25'48" north latitude and 66° 01'42" west longitude, is the largest body of water of the SJBES, comprising a surface area of 550 hectares. SJL has two major inputs of freshwater and one major tide-dependent water exchange transit channel (Suárez Channel). The SJL confronts serious environmental pollution; evidence of this is shown by the fishkill episodes occurring in this system causing frequent anoxic conditions, and by the presence of industrial and domestic inorganic and organic toxic chemical wastes. Metals are among the contaminants detected in sediments of the SJL with concentrations of lead and mercury as high as 548 µg/g and 4.9 µg/g (dw), respectively.

The CETR also has engaged in other environmental research projects with other government agencies such as the National Oceanic and Atmospheric Administration (NOAA), the Puerto Rico Water Authority, and the Puerto Rico Electric Power Authority. There is no doubt that the EPA-EPSCoR activities has advanced, strengthened, and enhanced environmental-related research in Puerto Rico through the development of the CERT.

Summary/Accomplishments (Outputs/Outcomes):

Objectives of Research Project 1

The main objective of this project was the development of a DNA electrochemical sensor for the detection of Escherichia coli and total coliforms. The research objectives were to: (1) identify gene sequence characteristics to E. coli; (2) synthesize and characterize polycations of ferrocene and 4-vinylpyridine; (3) study the physicochemical interaction of the polycations with Calf-Thymus (cT) DNA and oligonucleotide; and (4) construct an electrochemical sensor for E. coli using the electrochemical labels.

Probe Selection. Single-stranded DNA probes were designed based in the sequence of lac-Z and lamb genes. These probes were designed using DNAsis and HYBsimulator (Hitachi Software). From the analysis obtained from DNAsis and FASTA, only eight probe sequences met all the criteria and did not hybridize with any sequence reported in the European Molecular Biology Laboratory (EMBL) database.

Eleven probe sequences were tested using the DIG System (Boehringer Mannheim Corporation, Indianapolis, IN). The sequences were for lacZ: 5'accatgattacggattc3' (f), 5'ccatgaggacggattca3' (j), and 5'tgattacggattca3' (e). For lamB, the sequences were: 5'ctatgcacgttccggtat3' (c), 5'tatgcacgttccggtatt3' (b), 5'cttctaccaacgtcatga3' (h), 5'catgatgtacgtgggtat3' (i), 5'gggtatgtaccaggatat3' (g), 5'tgtacatcgggcgaatac3' (a), 5'tattcgcccgatgtacaa3' (d), and ccatctgggcaggtaagc3' (k). The probe was labeled according to manufacturer's instructions and hybridizes to colonies transferred to a nylon membrane. The bacteria tested as positive controls were: E. coli ATCC 23737, 23739, 12435; Enterobacter cloacae ATCC 13047, Citrobacter freundii ATCC 33128, and Klebsiella pneumoniae ATCC 13883. The negative controls were Salmonella typhi, and Proteus vulgaris ATCC 13315.

All experiments were conducted in triplicate. The results showed that probe a, b, c, d, and k (for lamB) and probe e for lacZ only hybridize with the positive control, except for E. coli ATCC 12435. In fact, E. coli 12435 did not hybridize with any of the probes tested.

Electroactive Label

Copolymers of Ferrocene and 4-Vinylpyridine. Polycations of different vinylferrocene and quaternized 4-vinylpyridine ratios were synthesized. The pyridine ring was quaternized with alkyl iodides of different alkyl groups. The effect of ionic strength, ferrocene to pyridine ratio and alkyl group size on the polycations binding ability with cT and 18-mer DNA, ds-(LamB1/cLamB1), cLamB1, ss-PNA analog of LamB1 (13-mer), and ds-(ss-PNA/cLAmB1) were studied. The results of the binding of polycations with cT-DNA showed that the binding constant decreases by one order of magnitude on increasing the ionic strength. According to Scatchard plots, the polycations contact the cT-DNA surface at least interacting with 9-10 nucleotides. Likewise, the binding constant decreases when the content of ferrocene decreases, showing that the oxidation of ferrocene contributes to the polycation interaction with the cT-DNA.

Studies also were conducted to determine the binding constant of the polycations with oligonucleotides using an 18-mer DNA; herein, single-stranded lamB1 (5'-CTA TGC ACG TTC CGG TAT-3'), and ss-lamB1 was one of the sequences identified in the Probe Selection experiments. Results showed that the Kb decreases slightly when the size of the alkyl group increases. The Scatchard plots indicated an interaction of three to five nucleotides with the polycations.

In all cases, there was a decrease on the peak current and potential for the EPA's Office of Solid Waste (OSW) voltammetry, indicating a strong interaction of cT-DNA and oligonucleotides with the polycations. This was further confirmed by melting temperature experiments, where an almost-zero baseline was observed up to the 110°C permitted by the instrument and the boiling temperature of the aqueous solvent. The redox potential change as the cT concentration increased is indicative of the contribution to the binding on the oxidation of the ferrocene center.

In experiments with ds-(ss-PNA/cLamB1), and ss-PNA, and the 75/25, 65/35, 50/50 vpy to vfc ratios results demonstrated the polycations interact with the ds-(ss-PNA/cLamB1). We also observed that the peak potential for the polycations was unaltered on the addition of ss-PNA. A decrease on the peak current also was observed but the magnitude was smaller than with the ds-(LamB1/cLamB1). This seems to indicate that the polycations can interact with the oligonucleotides through more than one type of interaction (i.e., not only by electrostatic interactions). Experimental studies and molecular modeling are underway to explain these results and to propose a model for the interaction of the polycation with the sequences at the molecular level.

Poly(Iron(II) tris(4-vinyl-4'-methyl-2,2'-bipyridine) Homopolymer, Chloride Salt. Poly-[Fe(vbpy)3]Cl2 (vbpy = 4-vinyl-4'-methyl-2,2'-bipyridine) was synthesized and characterized by high-performance liquid chromatography (HPLC), UV-Vis, Fourier Transform Infrared Spectroscopy (FTIR), and Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR). Its interaction with cT-DNA was studied by thermal denaturation measurement, UV-Vis spectroscopy, and electrochemistry. The results indicate a strong interaction of poly-[Fe(vbpy)3]Cl2 with ct-DNA. Furthermore, the interaction of the polymer with ct-DNA immobilized on a carbon paste electrode surface was investigated. This work showed that poly-[Fe(vbpy)3]Cl3 is an effective DNA carrier and a potential label for either a spectroscopic or an electrochemical DNA probe.

Sensor Construction

Initial experiments have been conducted to explore the adequacy of avidin-biotin interaction to immobilize DNA probes on electrode surfaces and to detect the hybridization step with the polycations. The results indicate that the avidin effectively blocked the gold surface and discriminates against positively charged molecules by repulsive electrostatic interactions. A biotinylated oligo was immobilized on the avidin-modified gold surface, taking advantage of the strong interaction between avidin and biotin.

The accumulation time of [Ru(phend)2dppz](PF6)2 on the Au/Avidin/Bio-S03 surface was investigated. A 10-minute accumulation time was enough to reach a limiting response. Ongoing studies are directed to construct a calibration curve to determine the validity of this approach for analytical determinations and to explore other immobilization strategies.

Objectives of Research Project 2

The main objective of this project was to obtain material and evaluate the chemical and toxicological effects of adsorbed compounds from atmospheric particulate matter in Guaynabo Basin onto different size of particulate matter (TSP, PM2.5, and PM10) from the Guaynabo Basin and a Reference Site. We characterized this PM through a year-long collection period from bases with heavy metal and carbon content. In addition, toxicological profiles (cytotoxicity, genotoxicity, and physiotoxicity) from extracts containing compounds of different polarities and size were generated.

Airborne PM2.5. Two PM sampling stations were established, one in an urban area (Guaynabo Basin) and the other at a reference site (Fajardo). PM2.5 were sampled at these stations from November 2000 to September 2001.

Significantly higher levels of PM2.5 were recorded at the Guaynabo Basin site when compared to Fajardo. This was expected for the Guaynabo Basin due to the anthropogenic contributions from industry. Guaynabo Basin levels ranged from 7.6 µg/m3 in April 2001 to 14.3 µg/m3 in July 2001. Fajardo concentrations fluctuated from 6.4 µg/m3 in April 2001 to 12.0 µg/m3 in June 2001. The annual average for PM2.5 in Guaynabo Basin was 11.6 ± 0.6 µg/m3, as compared to 8.5 ± 0.5 µg/m3 at Fajardo. When we examined the chemical composition of this PM using factor and cluster analyses (in the order to grouped variables) indicating common origin sources, we found the following: (1) all metal species analyzed, except Fe, were significantly higher in Guaynabo Basin compared to Fajardo; (2) average levels of PM2.5 in Guaynabo Basin were 11.6 µg/m3 versus 8.5 µg/m3 in Fajardo; (3) average levels of elemental carbon (EC) and organic carbon (OC) in Guaynabo Basin were 1.5 µg/m3 and 2.2 µg/m3, respectively; and (4) in Fajardo, EC levels were <0.14 µg/m3 and OC were <1 µg/m3. A very significant finding from our research is that Ni and V in the Guaynabo Basin exhibited very high concentrations (Ni = 17 ng/m3 and V = 40 ng/m3). This is evident when we compare concentrations obtained in Guaynabo Basin with those reported in different regions of the world (see Table 1).

Table 1. Heavy Metals and EC/OC Levels Found in PM2.5 From Different Regions of the World

Reference Site
PM2.5
EC
OC
As
Cd
Cu
Fe
Ni
Pb
V
Zn
Fang et al.(1999) THU (suburban),Taiwan
25.9
na
na
na
na
22
1,300
na
54
na
121
HKIT (rural),Taiwan
25.7
na
na
na
na
22
330
na
19
na
85
Singh et al.(2002) Downey (urban),Los Angeles
na
na
na
na
na
13
296
4.4
11
5.2
35
Riverside (inland),Los Angeles
na
na
na
na
na
4
170
1.9
4.1
4.1
13
Hien et al.(2001)* Ho Chi Minh,Viet Nam
na
na
na
2.5
na
2
261
na
79
7.8
245
Castanho et al.(2001) Sao Pablo (winter),Brazil
30.2
7.6
15.8
na
na
19
532
3.9
42
11.7
126
Sao Pablo (summer),Brazil
15.0
4.1
5.3
na
na
5
179
3.1
23
8.9
51
Wei et al.(1999) 4 cities,China
92
na
na
31
na
29
na
na
341
na
420
Marcazan et al. (2001) Milan,Italy
55
na
na
na
na
18
190
6.5
138
9
110
Chan et al.(1999) Brisbane,Australia
7.3
1.81
na
na
na
na
50
na
51
na
27
Querol et al.(2001) Castello,Spain
20
na
na
8.5
0.5
10
250
3.0
288
6
178
Wang et al.(2002)** Nanjing,China
na
na
na
na
na
22
17
4.5
13
2
73
Diociaiuti et al. (2001) Rome,Italy
31
na
na
na
1.6
na
322
1
124
3
34
Metal concentrations are given in ng/m3, PM2.5, and EC/OC are in µg/m3 (na = not analyzed).
* Particulate material analyzed was <2 µm.
** Water soluble elements.


Guaynabo Basin concentrations were several-fold higher than those reported on the table. Although PM2.5 concentrations at Guaynabo Basin were lower than those reported in these studies, the relative metal concentration for these elements was higher, suggesting a serious problem in the environmental quality of this area. The exposure levels of Ni and V are a serious health concern, because they are causative agents of acute lung injury and cancer. Multivariate analyses showed stronger relationships in Guaynabo Basin for Ni, V, PM2.5, Fe, As, Cu, and Pb. In Fajardo, the stronger associations were found for PM2.5, Fe, Cd, V, Ni, Pb, and Cu negatively.

Toxicological Data. Some extracts from PM exhibited a dose-dependent reduction on the B-globin DNA XL polymerase chain reaction (PCR) amplification signal, while others did not exhibit this effect. Significant reduction in DNA amplification was observed for both TSP polar (acetone) extracts in Guaynabo Basin. Polar TSP extracts from Guaynabo Basin were deleterious to DNA, but relative amplification increased at the highest extract concentration. This could be because of an increase in toxic components, which inhibits cytochrome P450s. This would become clearer as the chemical composition of these extracts are revealed.

The genotoxic results generated from the PM10 polar extracts sampled in parallel with the TSP. These showed significantly less DNA damage than the TSP polar extracts. This is contrary to what is expected, because the majority of the toxicity should be associated to smaller particle size. However, these effects are consistent with those obtained and presented in the physiotoxic section below.

The extract from the Guaynabo Basin station showed the greatest effect at a concentration of 25 µg/mL. Because of the possible enzyme inhibition as previously explained, it is important to evaluate dose response and DNA damage between 5 and 25 µg/mL. The data presented here are but a small fraction of the data required for a comprehensive analysis of the genotoxicological responses from the many scenarios available for PM. The concomitant chemical analysis of the substances present in these extracts are essential for a complete understanding of our genotoxicity, cytotoxicity, and physiotoxicity data.

Physiotoxic Effects. We also evaluated the effects of PM10 extracts on isolated rat tracheal smooth muscle responses to electrical field stimulation. Isolated rat tracheas from male Sprague Dawley rats were placed in an organ bath chamber and connected to a Grass FT.03 force transducer to measure the tension generated by the tracheal muscle.

The results show that dichloromethane (DCM) PM10 extracts obtained from the Guaynabo Basin area produced a significant depression of the sensitivity of the tracheal muscle to electrical field stimulation. DCM PM10 extracts obtained from Fajardo produced some depression of the sensitivity of the trachea to electrical field stimulation. The PM10 extracts did not produce any significant changes on the acetylcholine-induced contraction of the trachea.

The results indicate that PM10 extracts from both Guaynabo Basin and Fajardo are able to inhibit the contractile response of isolated rat tracheal smooth muscle to electrical field stimulation. In addition, PM10 extracts cannot induce any changes in the contractile response to acetylcholine. It seems that the probable effect of PM10 lies more closely related to regulatory elements of the smooth muscle cells present in the nervous plexus of the tissue or other regulatory areas, such as epithelium, which is the first tissue exposed to PM in the atmosphere.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 9 publications 4 publications in selected types All 3 journal articles
Type Citation Project Document Sources
Journal Article Perez UJ, Jimenez B, Delgado W, Rodriguez-Sierra CJ. Heavy metals in the false mussel, mytilopsis domingensis, from two tropical estuarine lagoons. Bulletin of Environmental Contamination and Toxicology 2001;66(2):206-213. R827780E01 (Final)
not available
Journal Article Reyes D, Rosario O, Rodriguez JF, Jimenez B. Toxic evaluation of organic extracts from airborne particulate matter in Puerto Rico. Environmental Health Perspectives 2000;108(7):635-640. R827780E01 (Final)
not available
Journal Article Rodriguez-Sierra CJ, Jimenez B. Trace metals in striped mojarra fish (Diapterus plumieri) from Puerto Rico. Marine Pollution Bulletin. 2002;44(10):1039-1045. R827780E01 (Final)
not available

Supplemental Keywords:

DNA biosensors, microorganism detection, Escherichia coli, coliforms, electroactive labels, electroactive polycations, ruthenium complexes., RFA, Health, Scientific Discipline, INTERNATIONAL COOPERATION, Air, Geographic Area, Water, HUMAN HEALTH, particulate matter, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Biochemistry, Health Effects, International, Drinking Water, ecological risk assessment, diagnostic tool, asthma, atmospheric particulate matter, detection, air toxics, atmospheric particles, epidemiology, human health effects, chemical byproducts, environmental risks, airborne particulate matter, Puerto Rico, peptide nucleic acids, air pollution, analytical methods, human exposure, PM, biosensors, drinking water treatment, drinking water contaminants, allergic response, drinking water system, biomarker

Progress and Final Reports:

Original Abstract