Necrotic and Inflammatory Changes on Acute Studies of Siam Leaf ( Chromolaena Odorata) Alcoholic Extract on Lead Induced Toxicity of Cardio-Pulmonary Morphology of Adult Albino Wistar Rats.

and were not treated with any of the extracts. Human exposure to lead and its compounds occurs mostly in lead related occupations with intraperitoneally. Groups C were fed and various sources like leaded gasoline, industrial administered intraperitoneally with Lead and processes such as smelting of lead and its treated with 800mg/Kg body weight of alcoholic combustion, pottery, boat building, lead- based extract respectively. Group D were also fed with painting, lead containing pipes, battery food, water, dose of Lead and alcoholic Extract recycling, grids, arm industry, pigments, printing of C. Odorata with 400mg/kg body weight and of books, etc. Though its widespread use has Group E were given food, water and a high dose discontinued in many countries of the world, it is of the alcoholic Extract which was 800mg/kg still used in many industries like car repair, body weight respectively. Both doses of extracts battery manufacturing and recycling, refining, were administered daily to the rats using oro-smelting, etc. Lead is a highly poisonous metal gastric tube in line with body weights. The affecting almost every organ in the body. Of all duration of this study was (21) twenty-one days the organs, the nervous system is the mostly after which the rats were sacrificed under affected target in lead toxicity, both in children sedation with chloroform. Organs were and adults. The toxicity in children is however of collected, fixed in 10% formal saline, processed, a greater impact than in adults. This is because sectioned and stained using Haematoxylin and their tissues, internal as well as external, are Eosin staining procedure at the Histopathology softer than in adults. Long-term exposure of laboratory of Hospital adults can result in decreased performance in (NDUTH), microscopically examined at X10 some tests of cognitive performance that and X40 magnifications for histopathological measure functions of the nervous Cardio-diagnosis. Results showed degenerative changes pulmonary system. Cardio-pulmonary effects of in the heart and lungs of some of the alcoholic leaf extract of Chromolaena odorata , a experimental adult albino wistar rats. plant commonly used for traditional medications, was evaluated using male Wistar albino rats. Acute and sub chronic toxicity was evaluated after 14 days of exposure. The LD50 changes. was 2154mg/kg. Alcoholic leaf extract of Chromolaena odorata used in traditional

properties like softness, malleability, ductility, has a relatively low melting point. When freshly poor conductibility and resistance to corrosion cut, lead is silvery with a hint of blue; it tarnishes seem to make difficult to give up its use. Due to to a dull gray color when exposed to air. Lead has its non-biodegradable nature and continuous use, the highest atomic number of any stable element its concentration accumulates in the and three of its isotopes are endpoints of major environment with increasing hazards (Hauser et nuclear decay chains of heavier elements. Lead al., 2017).
is a relatively unreactive post-transition metal. Its weak metallic character is illustrated by its Human exposure to lead and its compounds amphoteric nature; lead and lead oxides react occurs mostly in lead-related occupations with with acids and bases, and it tends to form various sources like leaded gasoline, industrial covalent bonds. Compounds of lead are usually processes such as smelting of lead and its found in the +2-oxidation state rather than the combustion, pottery, boat building, lead based +4-state common with lighter members of the painting, lead containing pipes, battery carbon group. Exceptions are mostly limited to recycling, grids, arm industry, pigments, printing organolead compounds. Like the lighter of books, etc. Though its widespread use has members of the group, lead tends to bond with discontinued in many countries of the world, it is itself; it can form chains and polyhedral still used in many industries like car repair, structures (Beeman et al., 2013). battery manufacturing and recycling, refining, smelting, etc. Lead is a highly poisonous metal Since lead is easily extracted from its ores, affecting almost every organ in the body. Of all prehistoric people in the Near East were aware of the organs, the nervous system is the mostly it. Galena is a principal ore of lead which often affected target in lead toxicity, both in children bears silver. Interest in silver helped initiate and adults. The toxicity in children is however of widespread extraction and use of lead in ancient a greater impact than in adults. This is because Rome. Lead production declined after the fall of their tissues, internal as well as external, are Rome and did not reach comparable levels until softer than in adults. Long-term exposure of the Industrial Revolution. In 2014, the annual adults can result in decreased performance in global production of lead was about ten million some tests of cognitive performance that tonnes, over half of which was from recycling. measure functions of the nervous system. Infants Lead's high density, low melting point, ductility and young children are especially sensitive to and relative inertness to oxidation make it useful. even low levels of lead, which may contribute to These properties, combined with its relative behavioural problems, learning deficits and abundance and low cost, resulted in its extensive lowered IQ (Hauser et al., 2017). use in construction, plumbing, batteries, bullets and shot, weights, solders, pewters, fusible Long-time exposure to lead has been reported to alloys, white paints, leaded gasoline, and cause anaemia, along with an increase in blood radiation shielding (Bretherick et al., 2016). pressure mainly in old and middle-aged people. Severe damage to the brain and kidneys, both in In the late 19th century, lead's toxicity was adults and children, were found to be linked to recognized, and its use has since been phased out exposure to heavy lead levels resulting in death. In of many applications. However, many countries pregnant women, high exposure to lead may cause still allow the sale of products that expose miscarriage. Chronic lead exposure was found to humans to lead, including some types of paints reduce fertility in males. Blood disorders and and bullets. Lead is a neurotoxin that damage to the nervous system have a high accumulates in soft tissues and bones; it damages occurrence in lead toxicity (Hauser et al., 2017).
the nervous system and interferes with the function of biological enzymes, causing Lead is a chemical element with the symbol Pb neurological disorders, such as brain damage and (from the Latin plumbum) atomic number 82. It behavioral problems. Lead also affects general is a heavy metal that is denser than most common health, cardiovascular, and renal systems materials. Lead is soft and malleable, and also (Betherick et al., 2017).
Pure lead has a bright, silvery appearance with a Identification hint of blue (Beiner et al., 2015). It tarnishes on The plant for this study, Chromolaena Odorata contact with moist air and takes on a dull was identified by Dr. Gideon Alade of the appearance, the hue of which depends on the Department of Pharmacognosy, Faculty of prevailing conditions. Characteristic properties Pharmacy, Niger Delta University Wiberforce of lead include high density, malleability, Island, Amassoma, Bayelsa state. ductility, and high resistance to corrosion due to Extraction of Chromolaena Odorata passivation. Bulk lead exposed to moist air forms The Plant material, Chromolaena odorata leaves a protective layer of varying composition. Lead were collected within the locality of Amassoma (11) carbonate (PbCO3) is a common in Southern Ijaw Local Government Area of constituent; the sulfate or chloride may also be Bayelsa state, Nigeria and were identified in the present in urban or maritime settings. This layer Department of Pharmacognosy, Faculty of makes bulk lead effectively chemically inert in Pharmacy, Niger Delta University, Nigeria. The the air. Finely powdered lead, as with many leaves of Chromolaena Odorata were dried metals, is pyrophoric and burns with a bluishet al under room temperature with further drying white flame (Bahrara ., 2006). using an oven and ground to fine powder using an electric grinder. Approximately 250 g of the Fluorine reacts with lead at room temperature, grounded sample was weighed using an forming lead (11) fluoride. The reaction with electronic weighing balance and dissolved in chlorine is similar but requires heating, as the 1000mL of absolute ethanol. This was properly resulting chloride layer diminishes the reactivity mixed and allowed to stand for 48 hours, after of the elements. Molten lead reacts with the which it was filtered using Whatman No. 1 filter chalcogens to give lead (11) chalcogenides et al paper. The filtrate was concentrated by heating in (Harbison ., 2015). a water bath at 40 ?C and the remaining solvent was removed in a rotary evaporator to produce Lead metal resists sulfuric and phosphoric acid but crude ethanolic extract of C. odorata. not hydrochloric or nitric acid; the outcome depends on insolubility and subsequent Animal Housing passivation of the product salt. Organic acids, such Twenty-five albino rats weighing between as acetic acid, dissolve lead in the presence of 150g+ 2.6-317g+ 3.5 were used for this study. oxygen. Concentrated alkalis will dissolve lead et al., These rats were obtained from the animal and form plumbites (Guruswamy 2000 month, the animals were allowed to acclimatize for two weeks. After the acclimatization period 25 rats were randomly divided into 5 groups with each group containing 5 rats per group (A-E).
body weight, pelleted growers feed (mash) Group A rats (control) were administered orally throughout the experiment for 30 days. with pelleted growers feed (mash) and water throughout the experiment.
Group D rats were administered orally with Chromolaena Odorata Extract (400mg/kg) for Group B rats (Positive control) rats were 30 days and injected intraperitoneally with lead intraperitoneally injected with Lead poison only (150ml/100kg) for 2 days and they were given (150ml/100mg) and given growers feed (mash) growers pellet feed (mash) and water and water for 2 days. throughout.
Group C were injected intraperitoneally with Group E rats were administered orally with lead poison (150ml/100mg) for 2 days, and then Chromolaena Odorata Extract and growers the extract was administered orally 800mg/kg pellet feed (mash) and water for 14 days.  Haematoxylin and Eosin staining procedure at December, 2021. It took a period of four weeks, the Histopathology laboratory of Niger Delta two weeks for environmental adaptaion and two Teaching Hospital (NDUTH) Okolobiri. weeks substance administration.

Staining Protocol
Sections were de-waxed in 2 changes of xylene Route of administration until all the wax was removed and sections were Lead was administered intraperitoneally. Siam hydrated in descending grades of alcohol starting leaf (Chromolaena Odorata) extract was from absolute, 80% alcohol, 70% alcohol, 50% administered orally using orogastric tube alcohol and finally water. Sections were stained with Harris Hematoxylin for 15 minutes and the Sample collection sections were rinsed in water. Tissue sections Upon completion of the weeks of substance were differentiated in 1% acid alcohol until the administration, the animals were sacrificed by nucleus retains the stain then the sections were administering chloroform as anaesthetic blued in Scot tap water for 2 minutes and substance. The rats were dissected to harvest the Counterstaining was done using Eosin for 2 liver which was fixed immediately with 10% minutes. Sections were dehydrated in ascending formalin.
grades of alcohol (50%, 70% 80% and absolute Tissue processing alcohol) and were clear and mounted using a The tissues were being processed according DPX and viewed using a microscope. Histological standard tissue processing,    Tabular description for group D. Tabular description for group E.

Discussion
Plate 1 represent animal in group A. Sections The Plate labelled 4.1 to 4.5 shows the morphology showed normal slides with transverse section, of the heart after staining with Haematoxylin and oblique section and the central nuclei Plate 1 was Eosin and viewed at 400X magnification. The plates used to represent the control group A. There was labelled 4:1 to 4.5 shows the morphology of the liver no histological alteration to section because the after the administration of the various treatments for control group was just fed with water and grower's 14 days. The slide labelled group A represents the pellet for 21 without administration of Lead and animals in the control group which were given feed aqueous Extract of Chromolaena Odorata. and water only. Those labelled group B are those to which lead was administered (0.6mg/kg), feed and Plate 2 slide shows degeneration of the water, the group labelled C were the animals given transverse muscle, vascular congestion. Plate 2 the extract (Chromolaena Odorata) (0.6mg/kg) was used to represent group B which was the lead(0.6mg/kg), feed and water. The group labelled positive control group which were given Lead D represent animals which were administered with a 0.6mg/kg. There were visible histological low dose of aqueous Extract (0.5mg/kg). lead presentation of enlargement of the transverse (0.6mg/kg), feed and water. Lastly the slide labeled muscles and vascular congestion. Which might E represents animals which were feed Chromolaena be as a result of the lead that was administered Odorata (0.9mg/kg).
which is in conjunction to the work done by (Hwang et al., 2001) and his result showed presence of Necrosis and large degeneration of the study carried out by; Omobowal et al. (2014). the heart muscles.
According to their study, lead caused a significant increase in blood lead level (BILL) Plate 3 slide shows degeneration of transverse a n d s e r u m m a l o n d i a l d e h y d e ( M D A ) muscles with area of Necrosis and congestion of concentration in rats treated with lead alone as blood vessels. Plate 3 was used to represent compared to the normal control and this result group C which was the group that was may be caused by effect of lead on heart administered with Lead intraperitoneally and structure. They showed that oxidative stress is an using an oral gavage tube a high dose of aqueous important mechanism of Lead-induced toxicity, Extract of Chromolaena Odorata was also imbalance and removal of reactive oxygen administered orally. This plate shows species in cellular structure causing damage to enlargement of transverse muscles and multiple membrane. membrane lipid and protein (Ahmed vascular congestion. It was observed also that the et al., Aziz et al., 2012). administration of the extract had no effect on the presence of Lead because there were visible Our finding is in agreement with previous reports degeneration of organs and tissues which is in (Kalia and Flora, 2005;Reddy et al., 2004) conjunction with a previous report (Enosakhare which shows that exposure to lead causes et al., 2018).
immunotoxicity and toxicity to organs, excessive intake of lead has been linked with Plate 4 slide shows degeneration of transverse cancer of the stomach, small intestine, large muscles(D) with areas of necrosis(N) and intestine, ovary, kidney, liver, lungs etc. congestion of blood vessels. Plate 4 was used to represent the group D which was administered Group C and D had no significant change in their with Lead and a low dose of the aqueous Extract values as compared to the control group of Chromolaena Odorata. This group showed (p>0.05). Group E also had no significant muscular degeneration and necrosis. The difference or increase in the values (p>0.05). administration of the Extract also had no effect on the Presence of Lead which may be as a result The only significant difference seen was in their of the low dose being administered in initial and final weight. A significant difference conjunction with "Evaluation of hepatotoxicity (p>0.05) was seen in the initial weight as with ethanolic Extract of Chromolaena Odorata" compared to their final weight. For group B (Imebong et al., 2018).
(Lead only) there was an increase in the weights of the animals after administration, which is Plate 5 slide shows normal morphology of the indicative for a giving them pseudo-weight. For Shows the Morphology of the heart after the groups C and D (leaf extract and Lead), it shows administration of the various treatments for 14 that the leaf extract had no effect on the Lead days. Slide shows normal morphology of the poison as the organs of the animals were hearth muscles, the transverse section(T), inflamed as seen in the plates above. oblique section (Q), the central nuclei (N). Plate 5 was used to represent group E which was Ingestion, thallation and dermal exposure of administered with only a high dose of aqueous Lead induces increase in respiratory disease and Extract of Chromolaena Odorata. The plate alteration of histological characterization in the showed a normal histology of the Muscle and cardiovascular tissues due to Pb in toxication, central nuclei which may be as a result of the (Jarrar et al., 2011;Al Naimi et al., 2011). dose administered which is in conjunction with ''Cardiovascular effect of Chromolaena Odorata The various photomicrograph of the Heart on adult albino wistar rat'' as previously reported sections in the various plates agrees with the (Asomugha et al., 2014). study carried out by Repetto et al. (2010). Similarly, Omobowal et al. (2014) in their study The various photomicrograph of the heart observed that exposure to lead caused a sections in the various plates agree however with significant increase in blood lead level (BILL) a n d

s e r u m m a l o n d i a l d e h y d e ( M D A )
Odorata) (0.6mg/kg) Lead (I mg/kg), feed and concentration in rats treated with lead alone as water. The group labelled D represent animals compared to the normal control and this result which with lead (0.5mg/kg), feed and water. may be caused by effect of lead on heart structure Lastly the slide labeled E represents animals damaged of hepatocyte and disturbance in heart which were feed Chromolaena Odorata rate. They showed that oxidative stress is an (0.9mg/kg). important mechanism of lead-induced toxicity, imbalance and removal of reactive oxygen Plate 6 represent animal in group A. Sections species in cellular structure causing damage to showed normal slides with Alveolar sac, membrane, membrane lipid and protein (Ahmed Alveolar duct and alveoli and intra alveolar et al., 2010;Aziz et al., 2012). septa. Plate 6 was used to represent the control group A. There was no histological alteration to Our finding is also in agreement with previous section because the control group was just fed reports (Kalia and Flora, 2005;Reddy et al., with water and grower's pellet for 21 without 2004) showing that exposure to lead causes administration of Lead and aqueous Extract of immunotoxicity and toxicity to organs, Chromolaena Odorata. excessive intake of lead has been linked with cancer of the stomach, small intestine, large Plate 7 slide shows enlargement of the alveoli, intestine, ovary, kidney, liver, lungs etc.
thickening of intra alveolar septa with presence of inflammatory cells. Plate 7 was used to The only significant difference seen was in their represent group B which was the positive control initial and final weight, a significant difference group which were given Lead 0.6mg/kg. There (p>0.05) was seen in the initial weight as were visible histological presentations of compared to their final weight. For group B (lead enlargement of the Alveoli, thickening of intra only) there was an increase in the weights of the alveolar septa with presence of inflammatory animals after administration, which is indicative cells. This might be as a result of the lead that was for an inflamed heart, giving them pseudo-administered which is in conjunction to the work weight. For groups C and D (leaf extract and done by (Wang et al., 2001) and his result showed lead), it shows that the leaf extract had no effect presence of Necrosis and large degeneration of on the lead poison as the organs of the animals the Alveoli duct. were inflamed as seen in the plates above.
Plate 8 slide shows enlargement of the alveoli, Ingestion, thallation and dermal exposure of lead thickening of the intra alveolar septa with induces increase in hepatic peroxidation and presence of inflammatory cells. Plate 8 was used alteration of histological characterization in the to represent group C which was the group that hepatic tissues due to Pb in toxication (Jarrar et was administered with Lead intraperitoneally al., 2011;Al Naimi et al., 2011). and using an oral gavage tube a high dose of aqueous Extract of Chromolaena Odorata was Discussion for Lungs also administered orally. This plate shows The Plate labelled 4.1 to 4.5 shows the enlargement of the alveoli, thickening of the intra morphology of the liver after staining with alveolar septa with presence of inflammatory hematoxylin and eosin and viewed at 400X cells marked by presence of Kupffer cells. It was magnification. The plates labelled 4:1 to 4.5 observed also that the administration of the show the morphology of the lung after the extract had no effect on the presence of Lead administration of the various treatments for 21 because there were visible degeneration of day. The slide labelled group A represents the organs and tissues which is in agreement with a animals in the control group which were given previous report (Hussein et al., 2011). feed and water only. Those labelled group B are those to which Lead was administered Plate 9 slide shows thickening of the intra alveolar (0.6mg/kg), feed and water, the group labelled C septa, necrosis of the alveoli with inflammatory were the animals given the extract (Chromolaena cells. Plate 9 was used to represent the group D which was administered with Lead and a low dose compared to their final weight. For group B of the aqueous Extract of Chromolaena Odorata. (Lead only) there was an increase in the weights This group showed thickening of the intra alveolar of the animals after administration, which is septa, Necrosis of the Alveoli which is marked with indicative for a giving them pseudo-weight. For inflammatory cells and presence of Kupffer cells.
groups C and D (leaf extract and Lead), it shows The administration of the Extract also had no effect that the leaf extract had no effect on the Lead on the Presence of Lead which may be as a result of poison as the organs of the animals were the low dose being administered in conjunction with inflamed as seen in the plates above. a work done by (Kouame et al., 2013).
Ingestion, thallation and dermal exposure of Plate 10 slide shows normal morphology of the Lead induces increase in respiratory disease and Alveoli, intra alveolar septa. Plate 10 was used to alteration of histological characterization in the represent group E which was administered with respiratory tissues due to Pb in toxication only a high dose of aqueous Extract of (Umukoro et al., 2006). Chromolaena Odorata. The plate showed a Conclusion normal histology of the Alveoli, intra alveolar This study shows that the oral administration of septa which may be as a result of the dose Lead for the period of 21days lead to the administered which is in conjunction with et al presence of inflammatory cells, enlargement of previous report (Phan ., 2001). the Alveoli, intra alveolar septa, Necrosis of the alveoli. It was also observed that the oral The various photomicrograph of the lung administration of Chromolaena Odorata at sections in the various plates agree however with various doses, both a high dose (800mg/kg) and a the previous studies carried out, lead caused a low dose of (400mg/kg). It had no effect on the significant increase in blood lead level (BILL) normal histology of the lungs. Further study a n d s e r u m m a l o n d i a l d e h y d e ( M D A ) should be carried out extending the duration of concentration in rats treated with lead alone as study and increasing the dosage of the extract compared to the normal control and this result given to observe if it can ameliorate the effect of may be caused by effect of lead on lung structure. lead poison. They showed that oxidative stress is an important mechanism of Lead-induced toxicity, imbalance Recommendation and removal of reactive oxygen species in Further study on lead toxicity on various organs cellular structure causing damage to membrane. et al should be carried out and detailed study on the membrane lipid and protein (Ahmad . 2010; Chromolaena Odorata et al effect of on biological Assi ., 2016). parameters and its anti-toxic effect on lead poison. Further study on the effect on Our finding is in agreement with a previous Chromolaena Odorata et al.
on lead using higher dose report (Phan , 2001) which indicated that should be carried out. exposure to lead causes immunotoxicity and toxicity to organs, excessive intake of lead has This study is meant to determine the effect of been linked with cancer of the stomach, small Lead toxicity to the heart and lungs of albino intestine, large intestine, ovary, kidney, liver, wistar rats and also to determine the histological lungs etc. effect of Chromolaena Odorata Extract on lead induced toxicity on the Heart and Lungs of Group C and D had no significant change in their albino wistar rats. values as compared to the control group (p>0.05). Group E also had no significant Declaration of Competing Interest difference or increase in the values (p>0.05).
The authors declared no potential conflicts of interest with respect to the research, authorship, The only significant difference seen was in their and/or publication of this article. initial and final weight, a significant difference (p<0.05) was seen in the initial weight as