Description

1 Inspiration

Heavy metal pollution is undoubtedly one of the most essential and crucial issues that people have to address, owing to its toxicity, longevity in the atmosphere, and the ability to build up in an organism’s body. For instance, heavy metals contaminate lakes leading to bioaccumulation in the organisms in the lake. With the development of industrialization, mining, and energy needs, heavy metals become the leading cause of occupational diseases. Statistics showed that an estimated amount of 12 million tons of crops has been lost in China due to heavy metal pollution. Many organisms are exposed to heavy metal pollution through the food chain. We aim to establish a method to accurately and effectively detect the existence of heavy metals in substances. By doing so, we can prevent heavy metals from hindering the biological growth of organisms and causing cancers in humans.

2 Background

2.1 Heavy metal pollution

Heavy metals are toxic metals with a density greater than 4.5 g/cm3, including gold, silver, copper, iron, mercury, lead, cadmium, etc. The drinking water quality standard in China stipulates that the limit for lead is 0.01 mg/L, 0.001 mg/L for mercury, 0.005 mg/L for cadmium, 0.01 mg/L for arsenic, 0.05 mg/L for chromium, and 1.0 mg/L for fluoride compound. Heavy metal pollutants enter hydrosphere, lithosphere and atmosphere impacting water, air, soil, etc, and stay for a long time in the environment. Heavy metals can enter the food chain, bioaccumulate and deteriorate the ecosystem in various ways, causing diseases and death in plants and animals. When heavy metals accumulate in the human body to a certain extent, they cause chronic poisoning. Metal toxicity is often associated with developmental neurotoxicity, inflammatory diseases, carcinogenesis, etc. (Fig.1)

Fig.1 Heavy metal source pathway and human exposure (Engwa G. A. et al., 2019)

2.2 Monitoring methods for heavy metal pollution

Heavy metal detection methods include atomic absorption spectrometry, atomic fluorescence method, high performance liquid chromatography, etc. These traditional detection methods often require expensive equipment and specialized skills, which presents a challenge to communities with limited resources. For example, many developing countries and rural areas lack proper equipment to monitor heavy metal pollution in water, soil and food. Therefore, an affordable and easy-to-use monitoring method is urgently needed to ensure environmental and food safety in these regions.

One approach is to apply sensitive organisms to visual monitoring of heavy metal pollution. Sensitive organisms can be plants, insects, fish, etc. They are very sensitive to heavy metal pollution in the environment and will show obvious changes when they are polluted. By observing and recording the responses of these organisms, we can quickly and easily assess the level of heavy metal pollution in the environment. This method has several advantages. Firstly, it is relatively simple and affordable, requiring no expensive equipment or specialized skills. Secondly, it can provide real-time monitoring results. Finally, it can promote public participation and awareness by making the pollution impact intuitively felt by ordinary people.

2.3 Mechanism of heavy metal response in organisms: MTF-1

Metal-responsive transcription factor-1 (MTF-1) is involved in the mechanism of heavy metal response in many organisms. MTF-1 is highly conserved: despite being found widely in mammals, insects, and other organisms, it possesses the same basic mechanisms throughout various species. MTF-1 is activated by heavy metals, hydrogen peroxide and other manners of oxidative stress (Lichtlen and Schaffner, 2001) and regulates the balance of metal ions and antioxidant reactions. It is an important factor for cells to adapt to stress conditions like exposure to heavy metals and hypoxia. MTF-1 plays an important role in the regulation of cell cycle, DNA repair, cell differentiation, and cellular apoptosis, and in physiological processes such as embryonic development and immune regulation.

In animals, when cells are exposed to the heavy metals, MTF-1 is activated and translocated into the cell nucleus. This process may occur through various mechanisms, including direct binding of heavy metal ions, generation of reactive oxygen species, and activation of relevant signaling pathways. MTF-1 can bind to heavy metals like zinc, copper, cadmium, mercury, and lead, through its zinc-finger domain located in its N-terminus. For most proteins, zinc-fingers primarily bind with heavy metal ions, especially having an affinity for zinc. Nevertheless, MTF-1 has 4 fingers (1-4) that mainly bind to cellular DNA and merely sense zinc, triggering a nuclear localization factor that imports the factor into the nucleus (Günther, et al. 2012), with the 2 zinc-binding fingers (5-6) hypothesized to serve as stabilization for the DNA-binding fingers (Andrews, 2001). MTF-1 has three activation domains located in its C-terminus. This specialization not only facilitates the recruits of transcriptional apparatus, but also allows for the regulation of its export from the nucleus and the inducibility by zinc (Günther, et al. 2012). Once in the nucleus, activated MTF-1 binds to DNA sequences known as metal response elements (MREs). These MREs are typically located in the promoter regions associated with genes involved in the response to heavy metals. Binding of MTF-1 to MREs activates the transcription of the corresponding genes, thereby promoting heavy metal-related gene expression. These genes include a range of metal-binding proteins, antioxidant enzymes, transporters, and detoxification enzymes, particularly a family of proteins named the Metallothionein (MT) which are important for heavy metal homeostasis and detoxification.

Fig.2 Pathways and downstream functions of the metal transcription factor MTF-1

3 Potential of Drosophila in monitoring heavy metal pollution

3.1 Characteristics of Drosophila melanogaster

Drosophila melanogaster (hereinafter Drosophila) more commonly known as fruit flies is one of the most studied eukaryotic organisms and has made fundamental contributions to different areas of biology. Drosophila has many advantages in the laboratory, such as a short life cycle, easy handling and inexpensive maintenance.

Drosophila typically has maroon or brick-red colored eyes and yellow-brown body. Male has black rings across its abdomen while female has a black patch and straight and flat wings. Adult fruit flies are only 2-3 mm long. Female is generally larger than male.

Fruit flies typically are attracted towards ripe fruits and vegetables, and appear in dark and humid places. Given the correct conditions, females lay up to 500 eggs on or near the surface of fermenting vegetables. The newborns take about one week to turn into adults during summer or in summer conditions. Then adults quickly mate and reproduce again.

Fig. 3 The life cycle of Drosophila melanogaster (Brischigliaro, et al., 2023)

3.2 Role of MTF-1 in heavy metal response in Drosophila

Similar to the mammalian MTF-1, the Drosophila homolog of MTF-1, dMTF-1, binds to MREs and activates the transcription of the genes that play a crucial role in heavy metal homeostasis and detoxification. Unlike mammalian MTF-1, dMTF-1 is resistant to low PH (6 to 6.5) and has different sensitivity to metals. Mammalian MTF-1 is best activated by zinc and cadmium, whereas due to aspects of heavy metal metabolism in Drosophila cells cadmium and copper are more potent inducers, however when transfected into mammalian cells, dMTF-1 responds to zinc like mammalian MTF-1. Research (Zhang, et al., 2001) suggests that MT binds to copper or cadmium to release zinc and trigger MTF-1 inversely.   

3.3 Advantages of Drosophila in heavy metal monitoring

Drosophila serves as an advantageous chassis organism for constructing a visual heavy metal biomonitoring system. The technology required for breeding Drosophila is both cost-effective and simplistic, necessitating only finger-shaped tubes and a solid medium with cornmeal as the primary component for culture (Wang et al., 2019). Drosophila exhibits high fecundity, with mature females capable of laying up to 100 eggs per day, and a short life cycle, requiring a minimum of 10 days to develop from egg to adult (Jennings, 2011). Additionally, Drosophila possesses distinct responsive traits that are easily observable in areas such as body and eye coloration, wing shape, and bristle length (Hales et al., 2015). Drosophila’s genetic background is relatively simple due to its diploid nature and small genome size of 180Mb, consisting of four chromosome pairs, three of which are autosomal. Genetic engineering techniques in Drosophila are well-established. Common gene targeting tools such as zinc finger nucleases, TALENs, and RNA-guided CRISPR/Cas9 have all been successfully adapted for use in Drosophila (Xu et al., 2019)

Fig. 4 Advantages of using Drosophila as a model organism (Anoar et al., 2021)

4 Our Project

4.1 Goal

We aim to use Drosophila as a chassis organism to develop a visual biomonitoring system for heavy metals. This system uses genetically engineered Drosophila lines. The user can grow the lines for 5-10 days in the environment and observe the sizes of eyes or wings to determine the level of heavy metal pollution. The system is of low cost and requires minimal equipment and professional personnel. Its social value is really high, since it can solve the problems of expensive testing equipment, complicated technology, and high cost in many remote areas

4.2 Overview

4.2.1 General design

Our project mainly uses genetic engineering and hybridization methods.

Firstly, we genetically engineered one Drosophila line to express MTF-1 driven by UAS (genotype: UAS-MTF-1) and another to express an apoptosis gene, Hid, driven by MRE (genotype: MRE-Hid). After hybridization, we obtained a Drosophila line with genotype: UAS-MTF-1/ MRE-Hid. In this line, MTF-1 can be activated by heavy metals, then bind to MRE and drive the expression of the downstream Hid gene.

In addition, we used two other Drosophila lines commonly used in lab, one with a GMR-GAL4 driver for compound eye development (genotype: GMR-GAL4) and the others with a Vg-GAL4 driver or a ptc-GAL4 driver for wing development (genotype: Vg-GAL4 or ptc-GAL4). These three lines express GAL4 during the development of eyes or wings. GAL4 protein is a class of transcription factors in yeast, which can bind to specific Upstream Active Sequences (UAS), and drive the expression of downstream UAS genes. We hybridized each of these three lines with the previously hybridized Drosophila line (genotype: UAS-MTF-1;MRE-Hid) and developed three final Drosophila lines (genotype 1: UAS-MTF-1;MRE-Hid/GMR-GAL4; genotype 2: UAS-MTF-1;MRE-Hid/Vg-GAL4; genotype 3: UAS-MTF-1;MRE-Hid/ptc-GAL4). These three lines are used for our visual biomonitoring system for heavy metals.

Fig. 5 Project design

4.2.2 Mechanism of our Drosophila monitoring system

In the Drosophila lines (genotype 1: UAS-MTF-1/ MRE-Hid/ GMR-GAL4; genotype 2: UAS-MTF-1/ MRE-Hid/Vg-GAL4; genotype 3: UAS-MTF-1;MRE-Hid/ptc-GAL4) we developed for the monitoring system, during the development of eyes or wings, GAL4 is expressed and activates the expression of MTF-1 via binding to UAS. With the increase of heavy metal concentration in the growing environment, an increased level of MTF-1 is activated and binds to MRE, leading to an upregulated expression of Hid in the developing eyes or wings. Since Hid causes cell apoptosis, the eye cells or wing cells of Drosophila show increased degree of apoptosis, resulting in decreased sizes of eyes or wings. So we can judge the degree of heavy metal pollution in the growing environment of Drosophila according to the changes of eye or wing sizes. The phenotype can be shown in the first generation of the line, so under heavy metal pollution, the abnormal development of eyes or wings can be detected in 5-10 days.

Fig. 6 Mechanism of the heavy metal monitoring system of Drosophila melanogaster (based on that of Brand & Perrimon, 1993)

4.3 Prospects and Advancements

The Drosophila visual monitoring system for heavy metals is poised to undergo further advancements and find broader applications in the future. In the future, we can expand the system's capabilities to detect multiple heavy metals simultaneously to make it more versatile. By expressing proteins in response to different heavy metal ions in different organs of Drosophila, the system can provide more comprehensive and detailed information about the composition of heavy metal contamination. This system also has the potential to be integrated with sensor technology to enable real-time data transmission, remote monitoring, and the development of predictive models for early warning systems.

In conclusion, our system offers a range of advantages, including sensitivity, real-time monitoring, cost-effectiveness, and environmental friendliness. Its application in environmental monitoring, occupational safety, agricultural and food safety, and public health management demonstrates its vast potential for positive impact. With ongoing research and future advancements, this system has the potential to revolutionize environmental monitoring practices, leading us towards a safer and healthier future

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