Pyrrolizidine Alkaloids:Toxicity,

Awareness and Regulations laid by

Government

Name : Farheen Zishan

Programme: MSc in Pharmaceutical Science

Module Code and Name : PP7102 Research

Skills
 Supervisor Name: Dr Eva Galante

Project proposal pro-forma (2019)

Abstract

Pyrrolizidine Alkaloidosis is a type of chronic toxicity due to the

presence of pyrrolizidine alkaloids (PAs) in many plants and herbs. PAs
are potent phytotoxins. Continuous long-term intake of plants or herbs
containing pyrrolizidine as a natural chemical ingredient causes three
major adverse effects in animal bodies i.e. hepatotoxicity, genotoxicity
and tumorigenicity. They are induced after metabolic digestion in liver,
initiating accumulation of harmful chemical substances called pyrroles as
a product. Pyrroles have the potential to inhibit cellular growth by Commented [1]: reference
inducing anti-mitotic effects in hepatocytes, the cells of the liver tissue
(Stegelmeier et al. 1999). In over 6000 plants, more than 300 to 600 types
of pyrrolizidine alkaloids have been identified. Food derived from plants
and herbs such as honey and other 200 known plant species contains PA
Herbal remedies based on herbs, weeds or plant ingestion may also cause
such toxicity. The result of this research is expected to deliver useful
insight into the fundamentals, causes, and characteristics of PAs toxicity,
including its role in hepatoxicity, tumorigenicity, and genotoxicity. The
regulations pertaining to PAs toxicity in UK and India are discussed, with
some beneficial information obtained from the comparison of both
policies. The literature gathered throughout the course of the study has
aided in creating a solid research base while highlighting basic ideas
regarding PAs toxicity (Chojkier).

Background information

Literature review

• Background area and scope to survey:

• Background:

Pyrrolizidine alkaloids are nitrogenous compounds which are made

of a five-carbon ring structure as shown in fig. 1. Although there is
no evident chemical structure of PAs with toxic characteristics, the
unsaturated PAs termed as PA 1, 2 are known to exhibit toxicity. Commented [2]: rephrase it, it is not clear. Vague,
more explation
PAs are present in above 600 plant species in minimal
concentrations. The first case of hepatic sinusoidal obstruction
syndrome in humans was identified in late 19th century due to
presence of pyrrolizidine N-oxides in their diets. Serious livestock
poisoning has also been recorded several times, due to the presence
of PAs in most the weeds of staple plants. Over three percent of
the world's flowering plants contain pyrrolizidine alkaloids.
Pyrrolizidine alkaloids are present as a metabolically active
ingredient in more than 30 potent phytotoxins which are found in
more than 200 species of plants, weeds and herbs.

Fig. 1 – structure of PAs

Hepatoxicity, Genotoxicity and Tumorigenicity

These toxic substances cause hepatoxicity, genotoxicity and
tumorigenicity in human systems. The main elements of the three
need to be studied to understand the ways in which they affect the
body. Metabolized and digested pyrrolizidine alkaloid induces
inhibition in the cellular growth as their main product is pyrrole.
Pyrroles through esterification is delivered into the liver cells. They
are metabolically reactive towards binding with nucleic acids and
proteins present in hepatocytes. Pyrroles binds to the DNA of
hepatocytes and induces anti-mitotic effects that inhibits the
multiplication and generation of new liver cells and initiates
formation of Megalocytes. After some time, Megalocytes are lysed
and replaced by fibrosis that causes scar formation in the liver's
tissue. After ingestion the esterified form of P.A enters the liver
cells, blocks the process of cellular mitosis by hijacked binding
with nucleic acids, D.N.A and proteins of the hepatocytes.
Afterwards, Megalocytes are formed, which eventually dies, and
liver fibrosis takes place. Hepatotoxicity could result in various
liver diseases and chronic syndromes as it doesn’t exhibit early
onset clinical symptoms. Liver disorders includes:

● Hepatic sinusoidal obstruction syndrome

 ● Hepatic fibrosis
● Hepatic cirrhosis
● Hepatic necrosis
● Hepatic failure

Genotoxicity is a part of genetics that pertains to the chemical

agent properties that disrupts genetic information in cells.
Genotoxicity causes mutations in the cell possibly leading to
cancer. It is defined that all genotoxic substances are not
mutagenic, even though all the mutagens can be classified as
genotoxic. DHA is directly affected by cell mutation, in turn
changing the organism’s somatic cells or the germ cells. These
conditions may concede to future generations. The PA poisoning
may directly or indirectly alter the structure of DNA. It can cause
carcinogenesis within the liver, directly affecting hepatic function
(Ma et al. 2018).

Tumorigenicity is known as the capability of cells which have been

cultured to create benign or malignant tumours that can
progressively grow. This condition directly affects the animal
system and is greatly dependent on the testing assay. If overdoses
of pyrrolizidine occur within the human body, it may cause
tumours to develop. The PA toxicity may cause abnormal growth
of cells at any part of the body’s system, resulting in a lump which
sometimes may or may not be evident immediately. The tumour
growths can be benign or malignant however, they may surface in
the animal body and indirectly affect functioning (Fu et al. 2004).

The effects of hepatoxicity, genotoxicity and tumorigenicity

therefore can prove to be quite dangerous. When exposed to the
harmful PA containing substances the animal bodies can be under
great threat. To eliminate the risks of toxicity further study into the
characteristics and causes of PAs needs to be carried out.

Scope of survey:

The accumulation of PAs is caused by continuous ingestion of

foods that contain the substance. Lack of awareness in the general
public could be causing chronic toxicity effects in individuals,
hence the need for appropriate chemical screening methods in
consumable goods. The survey of PAs and their toxic elements
searches various sources and records to evaluate previously
occurring instances of toxicity in individuals. Information
 pertaining to the presence of PA is naturally occurring food is
widely available and can be used beneficially to determine which
foods are most eminent subjects for screening. A variety of foods
are considered which require the methods of screening for toxic PA
substances and the basis for new and improved regulations for
lessening toxicity risks can be formed.

• Define subject: Commented [3]: not fully relevant and not well linked
to your background

Literature Review

It has been recently recognised that a list of foods that are

consumed daily may be contaminated by P.As. Poisoning or
serious side effects cannot occur from the determined amount of P.
As however the maximum allowed daily intake or levels is
exceeded. Primary data is acquired through collecting food
samples and testing them. Through the study, levels in foods
including honey and pollen, grains, milk, chicken eggs, meat,
salads, teas and condiments were observed (Ma et al. 2018).

Determining the sources and causes of this toxicity is a central part

of the study since P.A. is the most common type of poison to dwell
in plants and directly affects humans, wildlife, and livestock.
Although current regulations are present worldwide which inhibit
the use of P.A. containing plants and products derived from the
plants, more efforts must be made to increase awareness of the
matter. The harmful activities produced by P.A. toxicity such as
tumorigenicity is imperative in analysing the key strategies needed
to prevent the toxicity.

The in-depth analysis and insight into the causes and side effects of
P.A. toxicity and the activities it induces i.e. tumorigenicity,
genotoxicity, and hepatoxicity is necessary to improve conditions
regarding toxicity in consumable goods. The risks of P.A. toxicity
are increasing, and the assessed literature justifies the further study
into the concepts of toxicity and how to eliminate the potential
causes. Additionally, an increased global awareness is necessary by
amending or improving policies and regulations (Li et al. 2011).

The main goal of the comparison of two P.A. toxicity regulations is

to conclude whether hepatotoxicity and tumorigenicity is in fact
induced through metabolic activation relevant to long-term
ingestion of plants or herbs containing pyrrolizidine alkaloids.
World Health Organization in 1989 had issued health care and
safety guide book regarding awareness, health risks, preventive
measures, epidemics, toxic plant's regulations, guidelines and
standards for pyrrolizidine alkaloids. The preventive measures
included regulated and controlled use of toxic plants and edibles.

The European committee of medicinal and herbal products

(H.M.P.C) had introduced new analytical methods to detect P.A in
raw botanical products from low concentrations to trace amounts
as well. The H.M.P.C has regulated a guideline of maximum 0.35
micrograms/ litres of herbal product containing P.A for a
maximum 15 days only. The UK follows standards set by the
European Committee of Herbal and Medicinal Products
(H.M.P.C). There is no official test method for the analytical
quantification of toxic P.A.s. Due to this reason, the H.M.P.C
requested the European pharmacopoeia (E. P.) to take initiatives
towards the development of a standardized analytical method for
P.A.s in herbals medicinal products as a compulsory. The
following toxic P.A.s should be assessed by highly sensitive
analytical quantification tests introduced by the committee:

1. Jacobine-N-oxide
2. Senecionine-N-oxide
3. Erucifoline
4. Lasiocarpine
5. Seneciphylline
6. Erucifoline-N-oxide
7. Lasiocarpine-N-oxide
8. Seneciphylline-N-oxide
9. Europine
10. Lycopsamine
11. Senecivernine
12. Europine-N-oxide
13. Lycopsamine-N-oxide
14. Senecivernine-N-oxide
15. Heliotrine
16. Monocrotaline
17. Senkirkine
18. Heliotrine-N-oxide
19. Monocrotaline-N-oxide
20. Trichodesmine
21. Intermedine
22. Retrorsine
 23. Intermedine-N-oxide
24. Retrorsine-N-oxide

U.K Medicines agency (M.H.R.A) on 6th April, 2016 after

reviewing from German medicine agency's recently announced
threshold guidelines to lower the risk of P.A contaminations
announced the medicinal and herbal products registration holders
to allows a maximum threshold of 1.0 micro-grams of P.A
administration daily not to be exceeded for more than 3 years
(European Medicines Agency, 2016).

The Food Safety and Standards Authority of India (FSSAI) is

responsible for establishing and maintaining the food safety
regulations needed to keep consumables safe for the public. The
authority is run by Ministry of Health and Family Welfare and due
to the prevailing health concerns in the country, the monitoring of
naturally occurring foods which may be exposed to harmful
chemicals is necessary. India’s food safety authority does well to
comprehensively screen all the natural foods for any potential toxic
substances. The following foods are tested for toxic chemicals:

1. Grains
2. Fruits and Vegetables
3. Meats
4. Eggs
5. Fish
6. Milk and other dairy products
7. Eggs
8. Tea
9. Spices
10. Fish
11. Dry Fruits
12. Oils
13. Rice

There are a range of foods being tested, however only certain

chemical compounds are being searched for. The presence of P.A.
substances can affect most of these food products, but India’s
regulations do not account for the P.A. chemicals (FSSAI, 2019).
This shows the major discrepancy between the two major countries
in laying out the food standards for the country. If the effects of
P.A. on humans is not closely monitored, there could be great harm
 to the body. Certain strict regulations need to be set in place to
screen foods for specifically P.A. chemicals.

Identify and critically evaluate key published work and develop

rationale for your study Commented [4]: this is basically what has to be
discussed in your background!

• Link between concepts

• What information is missing? Commented [5]: this is meant to be the link withy your
aim. Has to be better developed

What is your research question?

How does PA toxicity cause hepatoxicity, genotoxicity and

tumorigenicity and which specific screening methods can help
reduce the occurrence of toxicity in animals? Which regulations
put forth by UK and India are useful for testing for toxic PA
substances and how can their comparison benefit in improving
health conditions?

• Hypothesis

It is predicted that PA toxicity’s effect on food consumption and

poisoning is largely significant since majority of the acquired data
and research points in the proposed direction. The policies for
controlling of substances that go into foods are the crucial factors
in preventing the poisoning by PA The regulations of India and UK
exhibit clear differences with the main discrepancies lying in the
background and status of the countries (Stegelmeier et al. 1999).

• Significance

The usefulness of the study carried out lies in the inherent need for
improving health and wellness for animals including humans
around the globe. In order to comprehend the instances of toxicity,
its causes, and methods of rectifying the issue, it is significant to
dig into the fundamentals of PA toxicity. How PA toxicity causes
adverse effects within animal bodies is a phenomenon which needs
considerably more study and while many instances of such toxicity
have been recorded, many undiscovered questions remain. A lot of
information regarding PAs is still unclear, starting with how many
substances relating to PAs occur naturally in consumable goods.
Diving into this study will not only aid in getting a clear
understanding of the working of PAs but can also prove useful to
 create future policies and regulations to prevent toxicity instances.
Therefore, the detailed analysis of the chemical components,
reactions, and characteristics of PA toxins is imperative to produce
a beneficial study. Commented [6]: more details

Aims

The aim of the study is to gain a clearer understanding than the existing
research around PA toxicity and link the concepts to current recorded
toxicity instances. A better understanding of the phenomenon and how it
affects animals’ systems is the key to creating effective methods of PA
screening in foods. It is also imperative to create a global awareness in
the general public regarding the toxic effects of plant and herb derived
edibles containing PAs to eliminate the risks of toxicity. All these factors
combined will give better insight into the causes of PA toxicity and allow
the creation of improved methods to lessen the associated risks (Tamariz
et al. 2018).

Methods and experimental design

Methodology

The systematic review and meta-analysis of the acquired data gives clear
insight into the proposed question. All major sources give purpose and
direction to the fact that PA toxicity is in fact a rather serious emerging
concern for various areas. It can affect the health of any group of people
pertaining to a specific location. The difference of the rules and
regulations of both countries was eminent and provided views into the
issue of health and awareness in specific areas.

The criteria for inclusion pertained to the fact that the source must
include information about at least one activity from hepatoxicity,
tumorgenicity and genotoxicity from PA The relevance of the source is
inherent to the study, with any unimportant data causing ambiguity in the
result, altering the hypothesis and its effectiveness. The criteria for
exclusion rejected any source which deviated from PA toxicity and
discussed or analysed irrelevant data for the issue (Yang et al. 2017).

The tool used for data collection was the sources found online and from
various journals and articles. This was directly incorporated within the
search strategy and the rationale behind the selected sources. The
extraction of data involved surveying through the collected resources and
determining which data from the entire set would be most advantageous
to the study.

The experimental design of the study included the appropriate selection

of resources, then the comprehensive analysis of each source according to
the information it provides. The data acquired during the study is
secondary data, each contributing uniquely to the research question. The
research is mainly of qualitative value since the data was not gathered,
rather obtained from other sources.

The research design that this study most closely corresponds with is the
case-study design since each source of information essentially acts as a
different case. The analysis and understanding of each source are
necessary to establish relations between each source and to appropriately
asses the contribution of each source to the study. Case study design
needs to be bias-free and should explain the phenomena expansively,
which in this study is the review of PA toxicity, the activities it causes
within animal bodies, and the comparison of the regulations made by
health enforcers in UK and India (Yang et al. 2016).

References

Chojkier, M. Hepatic sinusoidal-obstruction syndrome: toxicity of

pyrrolizidine alkaloids. Journal of Hepatology. 39(3), pp. 437 – 446.

European Medicines Agency. 2016. Public statement on contamination of

herbal medicinal products/traditional herbal medicinal products with
pyrrolizidine alkaloids. Committee on Herbal Medicinal Products.
London, UK.

Food Safety and Standards Authority of India (FSSAI). 2019. Food

Safety and Standards Regulations. [Online] Available at:
https://www.fssai.gov.in/home/fss-legislation/fss-regulations.html
[Accessed 4 May 2019].

Fu, P.P., et al. 2004. Pyrrolizidine Alkaloids—Genotoxicity, Metabolism

Enzymes, Metabolic Activation, and Mechanisms. Drug Metabolism
Reviews. 36(1), pp. 1-55.
Li, N., et al. 2011. Hepatotoxicity and tumorigenicity induced by
metabolic activation of pyrrolizidine alkaloids in herbs. Current Drug
Metabolism. 12(9).

Ma, C., et al. 2018. Determination and regulation of hepatotoxic

pyrrolizidine alkaloids in food: A critical review of recent research. Food
and Chemical Toxicology. 119(1), pp. 50-60.

Stegelmeier, B.L., et al. 1999. Pyrrolizidine alkaloid plants, metabolism

and toxicity. J. Nat Toxins. 8, pp. 95–116.

Tamariz, J., et al. 2018. Chapter One - Pyrrolizidine Alkaloids. The

Alkaloids: Chemistry and Biology. 80(1), pp. 1-314.

Yang, M., et al. 2017. First evidence of pyrrolizidine alkaloid N-oxide-

induced hepatic sinusoidal obstruction syndrome in humans. Archives of
Toxicology. 91(12), pp. 3913–3925.

Yang, M., et al. 2016. Cytotoxicity of pyrrolizidine alkaloid in human

hepatic parenchymal and sinusoidal endothelial cells: Firm evidence for
the reactive metabolites mediated pyrrolizidine alkaloid-induced
hepatotoxicity. Chemico-Biological Interactions. 243(1), pp. 119-126.